Methods of modulating and identifying agents that modulate intracellular calcium

ABSTRACT

Methods are provided for identifying agents that modulate intracellular calcium. Also provided are methods of modulating calcium within cells and methods of identifying proteins involved in modulating intracellular calcium.

RELATED APPLICATIONS

Benefit of priority is claimed to U.S. Provisional Application Ser. No.60/451,958, filed Mar. 4, 2003, entitled “Methods of Modulating andIdentifying Agents that Modulate Intracellular Calcium.” Wherepermitted, the subject matter and contents, including sequence listing,of this provisional application are incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The present invention relates to methods of identifying agents thatmodulate intracellular calcium. The invention further relates to methodsof modulating calcium within cells and treating disease by modulatingintracellular calcium. The invention also relates to methods ofscreening for candidate intracellular calcium-modulating proteins andnucleic acids encoding such proteins and methods of identifyingintracellular calcium-modulating proteins and nucleic acids encodingsuch proteins.

BACKGROUND OF THE INVENTION

Calcium plays a vital role in cell function and survival. For example,calcium is a key element in the transduction of signals into and withincells. Cellular responses to growth factors, neurotransmitters, hormonesand a variety of other signal molecules are initiated throughcalcium-dependent processes. Many proteins are activated by bindingcalcium and in turn affect other proteins in signal cascade mechanismsin cells. The normal basal concentration of free calcium in thecytoplasm of cells is about 50-100 nM whereas the extracellular calciumconcentration is typically about 2 mM. Therefore, intracellular calciumlevels and fluctuations thereof are tightly regulated by cells.

Calcium regulation by cells is accomplished through a variety ofmechanisms, some of which are associated with particular cell types. Forexample, excitable cells, such as muscle and nerve cells in whichcalcium signals are essential to functions including contraction andtransmission of nerve impulses, contain voltage-gated calcium channelsspanning the cell membrane. These channels respond to depolarization ofthe potential difference across the membrane and can open to permit aninflux of calcium from the extracellular medium and a rapid increase inintracellular calcium concentrations.

Nonexcitable cells, e.g., blood cells, fibroblasts and epithelial cells,as well as many excitable cells, contain channels that spanintracellular membranes and that can open to permit an influx of calciuminto the cytoplasm from calcium-storing organelles, such as theendoplasmic reticulum. One such intracellular ion channel is theinositol 1,4,5-triphosphate (IP₃) receptor located in the membrane ofthe endoplasmic reticulum. The IP₃ receptor functions as a ligand-gatedion channel that permits passage of calcium upon binding of IP₃ releasedthrough hydrolysis of membrane phospholipids by activated phospholipaseC (PLC). PLC can be activated through agonist binding to a surfacemembrane G protein-coupled receptor. Activation of the IP₃ receptorresults in the release of calcium stored in the endoplasmic reticuluminto the cytoplasm. Reduced endoplasmic reticulum calcium concentrationresulting from release of calcium therefrom provides a signal for influxof calcium from the extracellular medium into the cell. It appears thatthis influx of calcium does not rely on voltage-gated plasma membranechannels and does not involve activation of calcium channels by calcium.This calcium influx mechanism has been referred to as capacitativecalcium entry (CCE) or store-operated calcium entry (SOCE). The actualfactor that directly activates influx of calcium across the plasmamembrane in CCE is unknown, as is the identity of the molecule ormolecules that provide for mobilization of calcium across the plasmamembrane and into the cell.

Because of the vital role that calcium plays in cell function andsurvival, dysregulation of calcium in cells can have deleterious effectson cell structure and function. Alterations in intracellular calciumhomeostasis have been implicated in a variety of diseases.

There is a need, therefore, to elucidate the factors, structures andmechanisms involved in calcium regulation in cells, which may be targetsfor therapeutic intervention in diseases associated with calciumdysregulation. There is also a need for agents that modulateintracellular calcium and methods of identifying such agents as possibletherapeutic compounds for treatment of diseases associated with calciumdysregulation.

SUMMARY

Methods for identifying an agent that modulates intracellular calciumare provided. In one embodiment, the methods include the steps of:contacting one or more test cells or a portion thereof comprising one ormore proteins with a test agent, wherein the one or more proteins is/are(a) at least about 45% homologous to the protein encoded by Drosophilagene CG9126 and/or a stromal interacting molecule (STIM) or STIM-likeprotein over at least about 52% of the protein or (b) a portion of aprotein that is at least about 45% homologous to the protein encoded byDrosophila gene CG9126 and/or a stromal interacting molecule (STIM) orSTIM-like protein over at least about 52% of the protein; assessing theeffect(s) of the test agent on intracellular calcium; and identifying atest agent as an agent that modulates intracellular calcium if it has aneffect on intracellular calcium.

In another embodiment, the methods include the steps of: contacting oneor more test cells or a portion thereof comprising a stromal interactingmolecule (STIM) protein or portion thereof; assessing the effect(s) ofthe test agent on intracellular calcium; and identifying a test agent asan agent that modulates intracellular calcium if it has an effect onintracellular calcium.

In a further embodiment, the methods include the steps of: assessing theeffects of an agent on intracellular calcium, wherein the agentmodulates an activity of, an interaction of, the level of or binds to orinteracts with a protein that is at least about 45% homologous to theprotein encoded by Drosophila gene CG9126 and/or a stromal interactingmolecule (STIM) protein over at least about 52% of the protein; andidentifying a test agent as an agent that modulates intracellularcalcium if it has an effect on intracellular calcium.

In another embodiment, the methods include the steps of: assessing theeffects of an agent on intracellular calcium, wherein the agentmodulates an activity of, an interaction of, the level of or binds to orinteracts with a STIM or STIM-like protein; and identifying a test agentas an agent that modulates intracellular calcium if it has an effect onintracellular calcium.

In a further embodiment, the methods include the steps of: assessing theeffects of a test agent on (a) a protein that is at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) protein over at least about 52% ofthe protein or (b) a portion of a protein that is at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) protein over at least about 52% ofthe protein, wherein the test agent modulates intracellular calcium; andidentifying a test agent as an agent that modulates intracellularcalcium if it has an effect on a protein, or portion thereof, that is atleast about 45% homologous to the protein encoded by Drosophila geneCG9126 and/or a stromal interacting molecule (STIM) protein over atleast about 52% of the protein.

In another embodiment, the methods include the steps of: assessing theeffects of a test agent on a stromal interacting molecule (STIM) orSTIM-like protein, or portion thereof, wherein the test agent modulatesintracellular calcium; and identifying a test agent as an agent thatmodulates intracellular calcium if it has an effect on a STIM orSTIM-like protein, or portion thereof.

In methods of identifying an agent that modulates intracellular calcium,at least one of the one or more proteins can be STIM1 or a STIM2protein; a protein at least about 50% homologous, or at least about 62%homologous, to the protein encoded by Drosophila gene CG9126 over atleast about 77% of the encoded protein; a protein at least about 67%homologous to a mammalian STIM1 protein over at least about 86% of theprotein; a protein at least about 67% homologous to the amino acidsequence set forth as SEQ ID NO: 90 over at least about 86% of thesequence; or a STIM or STIM-like protein containing one or more of thefollowing domains: (a) a sterile “α-motif (SAM) domain comprising one ormore N-linked glycosylation sites and, optionally, an N-linkedglycosylation site in the amino acid sequence on either side of the SAMdomain, (b) a dibasic sequence of a proteolytic cleavage site, (c) anATP synthase B/B′ domain, (d) an ezxrin/radixin/moesin domain, and (e) adiacylglycerol kinase accessory domain. At least one of the one or moreproteins can be involved in, participate in and/or provide forstore-operated calcium entry. At least one of the one or more proteinscan be an ion transport protein or a component of an ion transportprotein complex.

In methods of identifying an agent that modulates intracellular calciumthat include a step of assessing the effects of an agent onintracellular calcium, the step can involve assessing the effect(s) ofthe test agent on store-operated calcium entry, the calcium level in anintracellular calcium store, the movement of an ion into, out of orwithin an intracellular calcium store, cytosolic calcium bufferingand/or resting cytosolic calcium levels. The step can involve assessingthe effect of test agent on intracellular and/or extracellular a) ionmovement, b) ion flux or c) ion levels of the one or more test cells.

In methods of identifying an agent that modulates intracellular calciumthat include a step of assessing the effects of a test agent on aprotein or portion thereof, the step can involve assessing binding orinteraction of the test agent with the protein or portion thereof;assessing the effect of the test agent on homotypic binding of theprotein or portion thereof or on binding of the protein or portionthereof to a second protein; assessing the effect of the test agent onthe level or size of the protein, or portion thereof, in a cell orportion thereof; assessing the effect of the test agent on the level orsize of nucleic acid encoding the protein, or portion thereof, in acell, or portion thereof; assessing the effect of the test agent on thelevel of expression of a nucleic acid sequence operatively linked to apromoter from a gene encoding the protein; assessing the effect of thetest agent on STIM protein, or a portion thereof, interaction with animmune system cell, or portion thereof; assessing the effect of the testagent on STIM-dependent augmentation of pre-B cell proliferation;assessing the effect of the test agent on STIM-dependent suppression oftumor cell growth; or assessing the effect of a test agent on Notchsignaling.

Also provided are systems that can be used, for example, in methods ofidentifying an agent or a molecule that modulates intracellular calcium.In one embodiment, the systems include: a cell, or portion thereof,comprising one or more heterologous proteins and/or heterologous nucleicacid encoding one or more proteins that is/are (a) at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) or STIM-like protein over at leastabout 52% of the protein or (b) a portion of a protein that is at leastabout 45% homologous to the protein encoded by Drosophila gene CG9126and/or a stromal interacting molecule (STIM) or STIM-like protein overat least about 52% of the protein; and an agent that provides forreduction of calcium levels in an intracellular calcium store.

In another embodiment, the systems include: a cell, or portion thereof,comprising one or more heterologous STIM of STIM-like proteins, or aportion thereof and/or heterologous nucleic acid encoding one or moreSTIM or STIM-like proteins, or a portion thereof; and an agent thatprovides for reduction of calcium levels in an intracellular calciumstore.

In a further embodiment, the systems include: a cell, or portionthereof, comprising one or more heterologous proteins and/orheterologous nucleic acid encoding one or more proteins that is/are (a)at least about 45% homologous to the protein encoded by Drosophila geneCG9126 and/or a stromal interacting molecule (STIM) or STIM-like proteinover at least about 52% of the protein or (b) a portion of a proteinthat is at least about 45% homologous to the protein encoded byDrosophila gene CG9126 and/or a stromal interacting molecule (STIM) orSTIM-like protein over at least about 52% of the protein; and a moleculeused in monitoring or measuring calcium and/or calcium movement.

In another embodiment, the systems include: a cell, or portion thereof,comprising one or more heterologous STIM or STIM-like proteins, or aportion thereof, and/or heterologous nucleic acid encoding one or moreSTIM of STIM-like proteins, or a portion thereof; and a molecule used inmonitoring or measuring calcium and/or calcium movement.

In such systems, the protein can be STIM1 or a STIM2 protein; a proteinat least about 50% homologous, or at least about 62% homologous, to theprotein encoded by Drosophila gene CG9126 over at least about 77% of theencoded protein; a protein at least about 67% homologous to a mammalianSTIM1 protein over at least about 86% of the protein; a protein at leastabout 67% homologous to the amino acid sequence set forth as SEQ ID NO:90 over at least about 86% of the sequence; or a STIM or STIM-likeprotein containing one or more of the following domains: (a) a sterile“α-motif (SAM) domain comprising one or more N-linked glycosylationsites and, optionally, an N-linked glycosylation site in the amino acidsequence on either side of the SAM domain, (b) a dibasic sequence of aproteolytic cleavage site, (c) an ATP synthase B/B′ domain, (d) anezxrin/radixin/moesin domain, and (e) a diacylglycerol kinase accessorydomain. The protein can be involved in, participate in and/or providefor store-operated calcium entry. The protein can be an ion transportprotein or a component of an ion transport protein complex. Alsoprovided are methods of identifying a molecule involved in modulatingintracellular calcium.

In one embodiment, the methods involve the step of: assessing the effectof a test molecule on intracellular calcium, wherein the test moleculeinteracts with a protein that is (a) at least about 45% homologous tothe protein encoded by Drosophila gene CG9126 and/or a stromalinteracting molecule (STIM) or STIM-like protein over at least about 52%of the protein or (b) a portion of a protein that is at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) or STIM-like protein over at leastabout 52% of the protein; and identifying a test molecule as a moleculeinvolved in modulating intracellular calcium if it has an effect onintracellular calcium.

In another embodiment, the methods involve the step of: assessing theeffect of a test molecule on intracellular calcium, wherein the testmolecule interacts with a STIM or STIM-like protein, or portion thereof;and identifying a test molecule as a molecule involved in modulatingintracellular calcium if it has an effect on intracellular calcium.

In the methods of identifying a molecule that modulates intracellularcalcium, the protein can be STIM1 or a STIM2 protein; a protein at leastabout 50% homologous, or at least about 62% homologous, to the proteinencoded by Drosophila gene CG9126 over at least about 77% of the encodedprotein; a protein at least about 67% homologous to a mammalian STIM1protein over at least about 86% of the protein; a protein at least about67% homologous to the amino acid sequence set forth as SEQ ID NO: 90over at least about 86% of the sequence; or a STIM or STIM-like proteincontaining one or more of the following domains: (a) a sterile “α-motif(SAM) domain comprising one or more N-linked glycosylation sites and,optionally, an N-liked glycosylation site in the amino acid sequence oneither side of the SAM domain, (b) a dibasic sequence of a proteolyticcleavage site, (c) an ATP synthase B/B′ domain, (d) anezxrin/radixin/moesin domain, and (e) a diacylglycerol kinase accessorydomain. The protein can be involved in, participate in and/or providefor store-operated calcium entry. The protein can be an ion transportprotein or a component of an ion transport protein complex. In methodsof identifying a molecule that modulates intracellular calcium thatinclude a step of assessing the effects of a test molecule onintracellular calcium, the step can involve assessing the effect(s) ofthe molecule on store-operated calcium entry, the calcium level in anintracellular calcium store, the movement of an ion into, out of orwithin an intracellular calcium store, cytosolic calcium bufferingand/or resting cytosolic calcium levels.

Also provided is a method of modulating intracellular calcium,comprising modulating in a cell, or portion thereof, one or moreproteins, or nucleic acid encoding one or more proteins, that is (are)at least about 45% homologous to the protein encoded by Drosophila geneCG9126 and/or a stromal interacting molecule (STIM) or STIM-like proteinover at least about 52% of the protein, thereby modulating intracellularcalcium in a cell or portion thereof.

In this method, intracellular calcium, for example, can be altered inthe cell or portion thereof. In embodiments of this method,store-operated calcium entry, calcium buffering, calcium levels in andintracellular calcium store and/or movement of calcium into, out of orwithin an intracellular calcium store can be altered in the cell orportion thereof.

In this method, modulating includes modulating the level of, expressionof, activity of or molecular interactions of one or more proteins ornucleic acid encoding one or more proteins. In certain embodiments,modulating includes increasing the level of, expression of, activity ofor molecular interactions of one or more proteins or nucleic acidencoding one or more proteins. In other embodiments, modulating includesreducing the level of, expression of, activity of or molecularinteractions of one or more proteins or nucleic acid encoding one ormore proteins.

The cells include mammalian cells, such as but are limited to rodentsand human cells. A portion of a cell in these methods includes a plasmamembrane, a cell organelle, an intracellular store or a membrane of acell organelle or intracellular store. In embodiments of these methods,at least one of the one or more proteins is a STIM1 or a STIM2 protein.In particular embodiments, the protein is at least about 50%, or atleast about 62%, homologous to the protein encoded by Drosophila geneCG9126 over at least about 77% of the encoded protein. The protein canbe at least about 67% homologous to a mammalian STIM1 protein over atleast about 86% of the protein. For example, the protein can be leastabout 67% homologous to the amino acid sequence set forth as SEQ ID NO:90 over at least about 86% of the sequence.

In other embodiments of these methods, the protein is a STIM orSTIM-like protein containing one or more of the following domains: (a) asterile α-motif (SAM) domain comprising one or more N-linkedglycosylation sites and, optionally, an N-linked glycosylation site inthe amino acid sequence on either side of the SAM domain, (b) a dibasicsequence of a proteolytic cleavage site, (c) an ATP synthase B/B′domain, (d) an ezxrin/radixin/moesin domain, and (e) a diacylglycerolkinase accessory domain. The protein can be one that is involved in,participates in and/or provides for store-operated calcium entry, andcan be, for example, an ion transport protein or a component of an iontransport protein complex. The protein can be a mammalian protein, suchas a rodent or human protein. In all methods, test molecules can be anymolecules, including proteins; the mammals include humans and rodents.

Also provided are isolated nucleic acid molecules that encode a rodentreference STIM1. In one exemplary embodiment, the isolated nucleic acidmolecules contain a sequence of nucleotides encoding rat STIM1 (SEQ IDNO: 97). In another exemplary embodiment, the isolated nucleic acidmolecules contain a sequence of nucleotides encoding hamster STIM1 (SEQID NO: 95). In one exemplary embodiment, the isolated nucleic acidmolecules contain a sequence of nucleotides encoding reference STIM1(SEQ ID NO: 52) or a portion thereof such as mature reference STIM1(amino acids 23-685 of SEQ ID NO: 52) or a polypeptide with no greaterthan 16 amino acid substitutions relative to the mature reference STIM1,which retains at least one biological activity of STIM1. In anotherembodiment, the isolated nucleic acid molecules contain a sequence ofnucleotides encoding a polypeptide hamster or rat STIM1 (SEQ ID NO: 96and 98).

In another embodiment, the isolated nucleic acid molecules contain asequence of nucleotides selected from among a) a nucleotide sequenceencoding rodent reference STIM1 extracellular domain; b) a nucleotidesequence encoding a polypeptide with no greater than 3 amino acidsubstitutions relative to a), which retains at least one biologicalactivity of STIM1 extracellular domain; c) a nucleotide sequenceencoding rodent reference STIM1 cytoplasmic domain; d) a nucleotidesequence encoding a polypeptide with no greater than 13 amino acidsubstitutions relative to c), which retains at least one biologicalactivity of STIM1 cytoplasmic domain; e) a sequence of nucleotidesencoding rodent reference STIM1 Glu-rich domain; and f) a nucleotidesequence encoding rodent reference STIM1 Pro-Ser rich domain.

In one embodiment, the isolated nucleic acid molecules are operativelylinked to a promoter of gene expression. In another embodiment, thenucleic acid molecules are contained in a vector. The nucleic acidmolecules or a vector containing the nucleic acid molecules can bepresent in a host cell.

Also provided are isolated oligonucleotides containing at least 17contiguous nucleotides of SEQ ID NO: 51, wherein the contiguousnucleotides include a position selected from among positions 15, 63, 69,84, 103, 108, 112, 120, 150, 183, 201, 207, 210, 213, 240, 300, 303,312, 330, 357, 402, 441, 474, 570, 621, 660, 697, 738, 783, 795, 861,873, 874, 895, 951, 1051, 1062, 1107, 1200, 1224, 1228, 1278, 1299,1392, 1395, 1452, 1580, 1652, 1654, 1675, 1747, 1749, 1173, 1854, 1855,1881, 1884, 1888, 1896, 2001 and 2025 of SEQ ID NO:51 or a correspondingposition in rodent reference STIM1 sequences as set forth in SEQ ID NOs:95 and 97. Also provided are isolated oligonucleotides that specificallyhybridizes to SEQ ID NO:51, but do not specifically hybridize to SEQ IDNO:3, SEQ ID NO:9 or SEQ ID NO:82.

Also provided are isolated STIM1 polypeptides and portions thereof. Inone embodiment, the polypeptides contain reference STIM1 (SEQ ID NO:52)or mature reference STIM1 (amino acids 23-685 of SEQ ID NO: 52) or anamino acid sequence with no greater than 13 amino acid substitutionsrelative to a rodent reference STIM1, such as set forth in SEQ ID NO:52,SEQ ID NO:96; and SEQ ID NO:98. In another embodiment, the polypeptidescontain a sequence of amino acids set forth in SEQ ID NO: 96 or SEQ IDNO: 98.

In another embodiment, the polypeptides contain a polypeptide selectedfrom among: a) rodent reference STIM1 extracellular domain; b) apolypeptide with no greater than 3 amino acid substitutions relative toa), which retains at least one biological activity of STIM1extracellular domain; c) rodent reference STIM1 cytoplasmic domain; d) apolypeptide with no greater than 13 amino acid substitutions relative toc), which retains at least one biological activity of STIM1 cytoplasmicdomain; e) rodent reference STIM1 Glu-rich domain; and f) rodentreference STIM1 Pro-Ser rich domain.

Also provided are isolated peptides containing at least 8 contiguousamino acids of SEQ ID NO: 52, wherein the contiguous amino acids includea position selected from positions 21, 35, 38, 292, 527, 551, 552, 583or 619 of SEQ ID NO: 52 or a corresponding position in SEQ ID NOS: 96and 98.

Also provided are antibodies that specifically bind to isolated rodentreference STIM1 peptides. In particular, the antibodies thatspecifically bind to a rodent reference STIM1 bind to the rodentreference STIM1 with at least 2-, 5, 10- or greater-fold affinity thanto a STIM1 from human or mouse. For example, provided is an antibodythat specifically binds to a polypeptide comprising the sequence ofamino acids set forth in SEQ ID NOS: 52, 96 or 98 but does notspecifically bind to a polypeptide comprising the sequence of aminoacids set forth in SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 85.

Provided are methods of modulating intracellular calcium by modulatingin a cell, or portion thereof, one or more proteins, or nucleic acidencoding one or more proteins, that is (are) at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) or STIM-like protein over at leastabout 52% of the protein.

The protein includes those that are involved in, participates in and/orprovides for store-operated calcium entry. The proteins include STIM1and STIM2 proteins, particularly STIM1. The methods include those inwhich least one of the one or more proteins is a STIM1 protein. Alsoincluded are methods in which at least one of the proteins is at leastabout 50% homologous, or at least about 62% homologous, to the proteinencoded by Drosophila gene CG9126 over at least about 77% of the encodedprotein. In other embodiments, the protein is at least about 67%homologous to a mammalian STIM1 protein over at least about 86% of theprotein.

In others, the protein is at least about 67% homologous to the aminoacid sequence set forth as SEQ ID NO: 90 over at least about 86% of thesequence. In certain embodiments, at least one or more proteins is aSTIM or STIM-like protein comprising one or more of the followingdomains: (a) a sterile α-motif (SAM) domain comprising one or moreN-linked glycosylation sites and, optionally, an N-linked glycosylationsite in the amino acid sequence on either side of the SAM domain, (b) adibasic sequence of a proteolytic cleavage site, (c) an ATP synthaseB/B′ domain, (d) an ezxrin/radixin/moesin domain, and (e) adiacylglycerol kinase accessory domain. Included among the proteins isan ion transport protein or a component of an ion transport proteincomplex. The protein can be a mammalian protein, including, for example,a rodent or human protein.

The cell or portion thereof in which the one or more proteins, ornucleic acid encoding one or more proteins, is modulated exhibitsaltered intracellular calcium. The altered intracellular calciumactivity can be altered store-operated calcium entry, altered calciumbuffering, altered calcium levels in an intracellular calcium storeand/or altered movement of calcium into, out of or within anintracellular calcium store, such as, for example, alteredstore-operated calcium entry. The portion of a cell can be plasmamembrane, a cell organelle, an intracellular store or a membrane of acell organelle or intracellular store. The cells include eukaryoticcells, such as mammalian cells. Mammalian cells include rodent and humancells.

Modulating can include modulating the level of, expression of, activityof or molecular interactions of one or more proteins or nucleic acidencoding one or more proteins. For example, modulating includesincreasing the level of, expression of, activity of or molecularinteractions of one or more proteins or nucleic acid encoding one ormore proteins. Alternatively, modulating can include reducing the levelof, expression of, activity of or molecular interactions of one or moreproteins or nucleic acid encoding one or more proteins.

Also provided are methods for identifying an agent that modulatesintracellular calcium, comprising: assessing the effects of a test agenton intracellular calcium of a test cell or portion thereof; andidentifying a test agent as an agent that modulates intracellularcalcium if it has an effect on intracellular calcium of the test cell.In these methods the test cell or a portion thereof comprises apolymorphic form of one or more proteins and/or a polymorphic form of agene or nucleic acid encoding one or more proteins with a test agent,wherein the one or more proteins is/are (a) at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) or STIM-like protein over at leastabout 52% of the protein or (b) a portion of a protein that is at leastabout 45% homologous to the protein encoded by Drosophila gene CG9126and/or a stromal interacting molecule (STIM) or STIM-like protein overat least about 52% of the protein; and intracellular calcium of the testcell differs from intracellular calcium of a cell, or portion thereof,that contains a wild-type form of the protein. Also provided are methodsfor identifying an agent that modulates intracellular calcium,comprising: assessing the effects of a test agent on intracellularcalcium of a test cell or portion thereof; and identifying a test agentas an agent that modulates intracellular calcium if it has an effect onintracellular calcium of the test cell. The test cell or a portionthereof comprises a polymorphic form of one or more STIM or STIM-likeproteins and/or a polymorphic form of a gene or nucleic acid encodingone or more STIM or STIM-like proteins; and intracellular calcium of thetest cell differs from intracellular calcium of a cell, or portionthereof, that contains a wild-type form of the protein(s).

In these methods, provided are embodiments where one or more of thefollowing differs in the test cell and a cell that contains a wild-typeform of the protein: store-operated calcium entry, cytosolic calciumbuffering, calcium level of an intracellular calcium store, movement ofcalcium into, out of or within an intracellular calcium store andresting cytosolic calcium level.

Also provided are methods for identifying an agent that modulatesintracellular calcium, by assessing the effects of a test agent onintracellular calcium of a test cell, or portion thereof, that exhibitscalcium dyshomeostasis; and identifying a test agent as an agent thatmodulates intracellular calcium if it has an effect on intracellularcalcium of the test cell. The test agent modulates an activity of, aninteraction of, the level of or binds to or interacts with a proteinthat is at least about 45% homologous to the protein encoded byDrosophila gene CG9126 and/or a stromal interacting molecule (STIM)protein over at least about 52% of the protein. Also provided aremethods for identifying an agent that modulates intracellular calcium,by assessing the effects of a test agent on intracellular calcium of atest cell, or portion thereof, that exhibits calcium dyshomeostasis; andidentifying a test agent as an agent that modulates intracellularcalcium if it has an effect on intracellular calcium of the test cell.The test agent modulates an activity of, an interaction of, the level ofor binds to or interacts with a STIM or STIM-like protein.

In these methods the test cell exhibits an alteration in one or more ofthe following relative to a substantially similar cell that does notexhibit calcium dyshomeostasis: store-operated calcium entry, cytosoliccalcium buffering, calcium levels of an intracellular calcium store,movement of calcium into, out of or within an intracellular calciumstore and resting cytosolic calcium levels. The test cell can be anysuitable cell, and is generally a eukaryotic cell, such as an immunesystem cell, a skin cell, a blood cell, a renal cell and a muscle cell.Exemplary of immune system cells are lymphocytes, such as T cells,including a T cells that exhibit a defect in activation. Also exemplaryof test cells are keratinocytes, including, for example, psoriatickeratinocytes. Other cells are mesangial cells, airway smooth musclecells. The test cells can be those that exhibit altered store-operatedcalcium entry relative to a substantially similar cell that does notexhibit calcium dyshomeostasis.

A protein in all of the methods includes those that are involved in,participates in and/or provides for store-operated calcium entry. Theproteins include STIM1 and STIM2 proteins, particularly STIM1. Themethods include those in which least one of the one or more proteins isa STIM1 protein. Also included are methods in which at least one of theproteins is at least about 50% homologous, or at least about 62%homologous, to the protein encoded by Drosophila gene CG9126 over atleast about 77% of the encoded protein. In other embodiments, theprotein is at least about 67% homologous to a mammalian STIM1 proteinover at least about 86% of the protein.

In others, the protein is at least about 67% homologous to the aminoacid sequence set forth as SEQ ID NO: 90 over at least about 86% of thesequence. In certain embodiments, at least one or more proteins is aSTIM or STIM-like protein comprising one or more of the followingdomains: (a) a sterile α-motif (SAM) domain comprising one or moreN-linked glycosylation sites and, optionally, an N-linked glycosylationsite in the amino acid sequence on either side of the SAM domain, (b) adibasic sequence of a proteolytic cleavage site, (c) an ATP synthaseB/B′ domain, (d) an ezxrin/radixin/moesin domain, and (e) adiacylglycerol kinase accessory domain. Included among the proteins isan ion transport protein or a component of an ion transport proteincomplex. The protein can be a mammalian protein, including, for example,a rodent or human protein.

In all of these methods the portion of the cell can include a plasmamembrane, a cell organelle, an intracellular store or a membrane of acell organelle or intracellular store.

In all methods, the test cells include mammalian cells, such as a rodentor human cell. In the methods, the protein can be encoded by nucleicacid that is heterologous to the cell. The test cell can be arecombinant cell and the gene or nucleic acid encoding the protein canheterologous to the cell. In these methods, the protein or at least oneof the one or more proteins is overexpressed in the test cell.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, by assessing the effects of a test agent onintracellular calcium of a test cell or portion thereof; and identifyinga test agent as an agent for treating or preventing a disease ordisorder if it has an effect on intracellular calcium of the test cell.The test cell is a cell of an animal that comprises a polymorphic formof one or more proteins and/or a polymorphic form of a gene, or portionthereof, or nucleic acid encoding one or more proteins with a testagent, wherein the one or more proteins is/are (a) at least about 45%homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) or STIM-like protein over at leastabout 52% of the protein or (b) a portion of a protein that is at leastabout 45% homologous to the protein encoded by Drosophila gene CG9126and/or a stromal interacting molecule (STIM) or STIM-like protein overat least about 52% of the protein. The cell comprises a polymorphic formof the one or more proteins and/or the polymorphic form of a gene, orportion thereof, or nucleic acid encoding the one or more proteinsexhibits calcium dyshomeostasis.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, by assessing the effects of a test agent onintracellular calcium of a test cell or portion thereof; and identifyinga test agent as an agent for treating or preventing a disease ordisorder if it has an effect on intracellular calcium of the test cell.The test cell is a cell of an animal comprising a polymorphic form ofone or more STIM or STIM-like proteins and/or a polymorphic form of agene, or portion thereof, or nucleic acid encoding one or more STIM orSTIM-like proteins. A cell comprising the polymorphic form of the one ormore proteins and/or the polymorphic form of a gene, or portion thereof,or nucleic acid encoding the one or more proteins exhibits calciumdyshomeostasis.

In these methods, one or more of the following is altered in a cellcomprising a polymorphic form of the one or more proteins or apolymorphic form of a gene, or portion thereof, or nucleic acid encodingthe one or more proteins: store-operated calcium entry, cytosoliccalcium buffering, calcium level of an intracellular calcium store,movement of calcium into, out of or within an intracellular calciumstore and resting cytosolic calcium level.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, by assessing the effects of a test agent on aphenotype of an organism; and identifying a test agent as an agent fortreating or preventing a disease or disorder if it has an effect on aphenotype of the organism. The test agent modulates an activity of, aninteraction of, the level of or binds to or interacts with a proteinthat is at least about 45% homologous to the protein encoded byDrosophila gene CG9126 and/or a stromal interacting molecule (STIM)protein over at least about 52% of the protein; and the organismcomprises one or more cells that exhibit calcium dyshomeostasis.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, by assessing the effects of a test agent on aphenotype of an organism; and identifying a test agent as an agent fortreating or preventing a disease or disorder if it has an effect on aphenotype of the organism. The test agent modulates an activity of, aninteraction of, the level of or binds to or interacts with a STIM orSTIM-like protein; and the organism comprises one or more cells thatexhibit calcium dyshomeostasis.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, by: assessing the effects of a test agent on aphenotype of an organism; and identifying a test agent as an agent fortreating or preventing a disease or disorder if it has an effect on aphenotype of the organism. The test agent modulates an activity of, aninteraction of, the level of or binds to or interacts with a proteinthat is at least about 45% homologous to the protein encoded byDrosophila gene CG9126 and/or a stromal interacting molecule (STIM)protein over at least about 52% of the protein; and the organismexhibits a phenotype associated with a disease or disorder that involvesor is characterized at least in part by calcium dyshomeostasis.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, by assessing the effects of a test agent on aphenotype of an organism; and identifying a test agent as an agent fortreating or preventing a disease or disorder if it has an effect on aphenotype of the organism. The test agent modulates an activity of, aninteraction of, the level of or binds to or interacts with a STIM orSTIM-like protein; and the organism exhibits a phenotype associated witha disease or disorder that involves or is characterized at least in partby calcium dyshomeostasis.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder, by: assessing the effects of a testagent on a phenotype of an organism; and identifying a test agent as anagent for treating or preventing a disease or disorder if it has aneffect on a phenotype of the organism. The test agent modulates anactivity of, an interaction of, the level of or binds to or interactswith a protein that is at least about 45% homologous to the proteinencoded by Drosophila gene CG9126 and/or a stromal interacting molecule(STIM) protein over at least about 52% of the protein; and the organismexhibits a phenotype associated with a disease or disorder selected fromthe group consisting of an immune system-related disease, a diseaseinvolving inflammation, a renal system disease, a neurodegenerativedisease, pain and liver disease.

Also provided are methods for identifying an agent for treating orpreventing a disease or disorder, by: assessing the effects of a testagent on a phenotype of an organism; and identifying a test agent as anagent for treating or preventing a disease or disorder if it has aneffect on a phenotype of the organism. The test agent modulates anactivity of, an interaction of, the level of or binds to or interactswith a STIM or STIM-like protein; and the organism exhibits a phenotypeassociated with a disease or disorder selected from the group consistingof an immune system-related disease, a disease involving inflammation, arenal system disease, a neurodegenerative disease, pain and liverdisease.

Diseases in the above methods include autoimmune diseases, diseases thatinvolve an immunodeficiency, diseases that involve glomerulonephritis.Diseases include, but are not limited to, psoriasis, asthma, andarthritis. The disease can involve exocrinopathy. Other diseases ordisorders include, but are not limited to, Sjogren's syndrome andneuropathic pain.

In all of the above methods, modulating one or more proteins, and/ornucleic acid encoding one or more proteins, can comprise exposing thecell, or portion thereof, to one or more agents that modulate the one ormore proteins, and/or a gene or nucleic acid encoding the one or moreproteins. The one or more agents can modulate the level, expression,functioning, molecular interactions and/or activity of the one or moreproteins, and/or a gene or nucleic acid encoding the one or moreproteins.

Also provided are methods for preventing a disease or disorder, bymodulating in a subject having a disease or disorder, or at risk fordeveloping a disease or disorder, one or more proteins, and/or a gene ornucleic acid encoding one or more proteins, that is (are) at least about45% homologous to the protein encoded by Drosophila gene CG9126 and/or astromal interacting molecule (STIM) or STIM-like protein over at leastabout 52% of the protein. The disease or disorder involves, or ischaracterized at least in part by: (1) altered intracellular calcium,altered intracellular calcium regulation or calcium dyshomeostasis ordysregulation and/or (2) an alteration or defect in, or aberrantfunctioning of, a cellular process which relies on or is regulated byintracellular calcium. For example, the disease or disorder can involve,or is characterized at least in part, by altered store-operated calciumentry, altered calcium buffering, altered calcium levels in anintracellular calcium store, and/or altered movement of calcium into,out of or within an intracellular calcium store, such as, for example,by altered store-operated calcium entry. In these methods, modulatingincludes modulating the level of, expression of activity of or molecularinteractions of one or more proteins and/or a gene or nucleic acidencoding one or more proteins. Modulating includes increasing the levelof, expression of, activity of or molecular interactions of one or moreproteins and/or a gene or nucleic acid encoding one or more proteins.Modulating also includes reducing the level of, expression of, activityof or molecular interactions of one or more proteins and/or a gene ornucleic acid encoding one or more proteins. In these methods, at leastone of the one or more proteins is a STIM1 or a STIM2 protein. Includedis a protein that is at least about 50% homologous to the proteinencoded by Drosophila gene CG9126 over at least about 77% of the encodedprotein or at least about 62% homologous to the protein encoded byDrosophila gene CG9126 over at least about 77% of the encoded protein.The protein can be least about 67% homologous to a mammalian STIM1protein over at least about 86% of the protein, including a protein thatis at least about 67% homologous to the amino acid sequence set forth asSEQ ID NO: 90 over at least about 86% of the sequence. The protein canbe a STIM or STIM-like protein comprising one or more of the followingdomains: (a) a sterile α-motif (SAM) domain comprising one or moreN-linked glycosylation sites and, optionally, an N-linked glycosylationsite in the amino acid sequence on either side of the SAM domain, (b) adibasic sequence of a proteolytic cleavage site, (c) an ATP synthaseB/B′ domain, (d) an ezxrin/radixin/moesin domain, and (e) adiacylglycerol kinase accessory domain. Exemplary of the proteins is aprotein that is involved in, participates in and/or provides forstore-operated calcium entry. The protein can be an ion transportprotein or a component of an ion transport protein complex. The proteinsinclude human proteins.

In these methods, modulating one or more proteins, and/or a gene ornucleic acid encoding one or more proteins, can include the steps of:administering to the subject one or more agents that modulate the one ormore proteins, and/or a gene or nucleic acid encoding the one or moreproteins. The one or more agents can modulate the level, expression,functioning, molecular interactions and/or activity of the one or moreproteins, and/or a gene or nucleic acid encoding the one or moreproteins. An agent can bind to or interact with one of the proteinsand/or a gene or nucleic acid encoding one of the proteins. Exemplaryagents are selected among: proteins, peptides, antibodies or fragmentsthereof and nucleic acids. Other exemplary agents are DNA or RNA.

The one or more proteins, and/or a gene or nucleic acid encoding one ormore proteins is/are modulated in cells, which include: immune cells,fibroblasts, skin cells, blood cells, renal cells, muscle cells,exocrine cells and secretory cells, such as, but are not limited to,lymphocytes, keratinocytes, mesangial cells, airway smooth muscle cells,lung cells, salivary gland cells and lacrimal gland cells.

The disease or disorder includes: immune system-relateddiseases/disorders, diseases/disorders involving inflammation,glomerulonephritis, hepatic diseases/disorders, renaldiseases/disorders, neurodegenerative diseases/disorders, aging-relateddiseases/disorders, sensitivity to pain or touch, chronic obstructivepulmonary disease, rheumatoid arthritis, inflammatory bowel disease,neuroinflammatory diseases, Alzheimer's disease, amytrophic lateralsclerosis, traumatic brain injury, multiple sclerosis, vasculitis,inflammatory bowel disease, dermatitis, osteoarthritis, inflammatorymuscle disease, allergic rhinitis, vaginitis, interstitial cystitis,scleroderma, osteoporosis, eczema, allogeneic or xenogeneictransplantation, graft rejection, graft-versus-host disease, lupuserythematosus, type I diabetes, pulmonary fibrosis, dermatomyositis,thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis), myastheniagravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsinghepatitis, primary biliary cirrhosis, allergic conjunctivitis, hepatitisand atopic dermatitis, such as but are not limited to, asthma, Sjogren'ssyndrome, Scott syndrome, glomerulonephritis, autoimmunediseases/disorders and immunodeficiency-related diseases/disorders. Thedisease or disorder includes a primary immunodeficiency or a severecombined immunodeficiency.

Also provided are methods of identifying an agent that modulatesintracellular calcium. The methods include contacting, with a testagent, one or more test cells overexpressing a mammalian STIM1 proteinor a portion thereof that retains at least one biological activity of amammalian STIM1 protein, assessing the effect of the test agent onintracellular calcium, and identifying a test agent as an agent thatmodulates intracellular calcium if it has an effect on intracellular. Inanother embodiment, the method includes contacting one or more testcells with a test agent, wherein the test agent modulates an activityof, modulates an interaction of or modulates the level of, or binds toor interacts with a mammalian STIM1 protein or a portion thereof thatretains at least one biological activity of a mammalian STIM1 protein,assessing the effect of the test agent on intracellular calcium, andidentifying a test agent as an agent that modulates intracellularcalcium if it has an effect on intracellular calcium. In anotherembodiment, the method includes assessing the effect of the test agenton an activity of, an interaction of or the level of a mammalian STIM1protein or a portion thereof that retains at least one biologicalactivity of a mammalian STIM1 protein, or on binding to or interactionwith mammalian STIM1 protein or a portion thereof that retains at leastone biological activity of a mammalian STIM1 protein, wherein the testagent modulates intracellular calcium, and identifying an agent thatmodulates the STIM1 protein or portion thereof if it has an effect on anactivity of, an interaction of or the level of STIM1, or binds to orinteracts with the STIM1 protein or portion thereof.

In the methods herein the test cell can be an immune cell. In themethods, the STIM1 can be human STIM1. The step of assessing in themethods herein can include assessing the effect(s) of the test agent ona calcium entry-mediated event. In one embodiment of the methods, thetest agent that modulates intracellular calcium modulates a calciumentry-mediated event. In another embodiment, the calcium entry-mediatedevent is selected from the group consisting of cytokine expression,cytokine secretion, NFAT dephosphorylation, NFAT nuclear localization,NFAT transcriptional activation, calcineurin phosphatase activity, andinflammatory mediator release. The cytokine can be IL-2 or TNFα. Theinflammatory mediator can be β-hexosaminidase.

Also provided herein are methods of identifying an agent that modulatescytokine expression or secretion. The methods include contacting, with atest agent, one or more test cells overexpressing a mammalian STIM1protein or a portion thereof that retains at least one biologicalactivity of a mammalian STIM1, assessing the effect of the test agent oncytokine expression or secretion; and identifying a test agent thatmodulates cytokine expression or secretion. In one embodiment, themethod includes contacting one or more test cells with a test agent,wherein the test agent modulates an activity of, modulates aninteraction of or modulates the level of, or binds to or interacts witha mammalian STIM1 protein or a portion thereof that retains at least onebiological activity of a mammalian STIM1 protein, assessing the effectof the test agent on cytokine expression or secretion; and identifying atest agent that modulates cytokine expression or secretion. In themethods the cytokine can be interleukin-2 (IL-2). In the methods, thetest cell can be an immune cell.

Also provided herein are systems. In one embodiment, the system includesa cell, or portion thereof, overexpressing one or more mammalian STIM1proteins or a portion thereof that retains at least one biologicalactivity of a mammalian STIM1 protein, and a molecule used inmonitoring, measuring or detecting a calcium-entry mediated event. Inthe systems, the STIM1 can be human STIM1. In the systems, the cell canbe an immune cell. In another embodiment, the system includes a cell, orportion thereof, containing a heterologous nucleic acid moleculecomprising one or more mammalian STIM1 nucleic acid molecules or aportion thereof, wherein the expression of STIM1 in the cell or portionthereof is reduced, and a molecule for monitoring, measuring ordetecting a calcium-entry mediated event. In the systems, theheterologous nucleic acid can be an interference nucleic acid moleculesuch as a double-stranded RNA (dsRNA), an antisense RNA, siRNA or RNAi.

FIGURE DESCRIPTIONS

FIG. 1 shows exemplary calcium entry-mediated events in immune cells.For example, as depicted, receptor activation leads to depletion of ERcalcium stores, which activates capacitative calcium entry throughI_(crac) channels. Calcium entry activates calmodulin-dependent enzymesincluding the serine phosphatase calcineurin, which leads to release ofinflammatory mediators in mast cells. Activation of calcineurin alsoleads to dephosphorylation, nuclear translocation and activation oftranscription factors such as NFAT. NFAT activation results inexpression and release of cytokines and T cell proliferation.Abbreviations: CCE, calcium capacitative entry; I_(CRAC), calciumrelease activated calcium channel current; PLCγ, phospholipase C gamma;IP₃, Inositol 1,4,5-triphosphate; SERCA, sarcoplasmic/endoplasmicreticulum calcium ATPase; DAG, diacyl glycerol; ER, endoplasmicreticulum; CaM, Calmodulin; NFAT, nuclear factor of activated T cells.

FIG. 2 is a graph showing fluorescence vs. time as measured in HEK293cells which had been loaded with a fluorescent indicator and subjectedto conditions designed to assess store-operated calcium entry into thecell (see EXAMPLES for details of the methods). The curve labeled as“STIM1” reflects fluorescence levels in a cell in which STIM1 proteinand mRNA expression had been reduced to non-detectable levels usingsiRNA-based interference methods described herein. The curve labeled“Control” reflects fluorescence levels in a cell that had been treatedwith control RNAs as described herein. At time “0”, 1 μM thapsigarginwas added to the essentially calcium-free medium. The increase influorescence upon addition of thapsigargin reflects an increase(followed by decrease) in calcium levels in the cytosol due to releaseof calcium from intracellular calcium stores. At about 16 minutes, 1.8mM calcium was added to the medium. The increase in fluorescence uponaddition of calcium to the medium reflects store-operated calcium entryinto the cytosol.

FIG. 3 is a graph showing fluorescence vs. time as measured in HEK293cells which had been loaded with a fluorescent indicator and subjectedto conditions designed to assess store-operated calcium entry into thecell (see EXAMPLES for details of the methods). The curve labeled as“STIM1” reflects fluorescence levels in a cell in which STIM1 proteinand mRNA expression had been reduced to non-detectable levels usingsiRNA-based interference methods described herein. The curve labeled“Control” reflects fluorescence levels in a cell that had been treatedwith control RNAs as described herein. At time “0”, 300 μM methylcholinewas added to the essentially calcium-free medium. The increase influorescence upon addition of thapsigargin reflects an increase(followed by decrease) in calcium levels in the cytosol due to releaseof calcium from intracellular calcium stores. At about 5 minutes, 1.8 mMcalcium was added to the medium. The increase in fluorescence uponaddition of calcium to the medium reflects calcium influx into the cellthat includes store-operated calcium entry.

FIG. 4 is a graph showing fluorescence vs. time as measured in HEK293cells which had been loaded with a fluorescent indicator and subjectedto conditions designed to assess store-operated calcium entry (SOCE)into the cell (see EXAMPLES for details of the methods). The curveslabeled as “STIM1” (dotted line) reflects fluorescence levels in a celloverexpressing STIM1 (HEK[STIM1]). The curves labeled “Control” (solidline) reflect fluorescence levels in a control HEK293 cell. At time “0”,either DMSO (Panel A) or 1 μM thapsigargin (Panel B) was added to theessentially calcium-free medium. At about 15 minutes, 1.8 mM calcium wasadded to the medium. Overexpression of STIM1 results in constitutiveactivation of Ca²⁺ entry, enhanced thapsigargin-activated Ca²⁺ entry,and a reduction in the amplitude and kinetics of thethapsigargin-mediated release of calcium from the internal stores.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, patent applications,publications and published nucleotide and amino acid sequences (e.g.,sequences available in GenBank or other databases) referred to hereinare incorporated by reference. Where reference is made to a URL or othersuch identifier or address, it is understood that such identifiers canchange and particular information on the internet can come and go, butequivalent information can be found by searching the internet. Referencethereto evidences the availability and public dissemination of suchinformation.

As used herein, “calcium homeostasis” refers to the maintenance of anoverall balance in intracellular calcium levels and movements, includingcalcium signaling, within a cell.

As used herein, “calcium dyshomeostasis” refers to altered, abnormal orimpaired calcium homeostasis. For example, calcium dyshomeostasis can beimbalances or disturbances in intracellular calcium levels or movementssuch as may result from altered calcium regulation in a cell.

As used herein, “intracellular calcium” refers to calcium located in acell without specification of a particular cellular location. Incontrast, “cytosolic” or “cytoplasmic” with reference to calcium refersto calcium located in the cell cytoplasm. Intracellular calcium can befree calcium and/or bound calcium.

As used herein, an “effect on intracellular calcium” is any alterationof any aspect of intracellular calcium, including but not limited to, analteration in intracellular calcium levels, calcium location andmovement of calcium into, out of or within a cell or intracellularcalcium store or organelle. For example, an effect on intracellularcalcium can be an alteration of the properties, such as, for example,the kinetics, sensitivities, rate, amplitude, and electrophysiologicalcharacteristics of calcium flux or movement that occurs in a cell orportion thereof. An effect on intracellular calcium can be an alterationin any intracellular calcium-modulating process, including,store-operated calcium entry, cytosolic calcium buffering, and calciumlevels in or movement of calcium into, out of or within an intracellularcalcium store. Any of these aspects can be assessed in a variety of waysincluding, but not limited to, evaluation of calcium or other ionparticularly cation) levels, movement of calcium or other ion(particularly cation), fluctuations in calcium or other ion(particularly cation) levels, kinetics of calcium or other ionparticularly cation) fluxes and/or transport of calcium or other ionparticularly cation) through a membrane. An alteration can be any suchchange that is statistically significant. Thus, for example ifintracellular calcium in a test cell and a control cell is said todiffer, such difference can be a statistically significant difference.

As used herein, “calcium entry-mediated event” is an effect onintracellular calcium, and refers to any cellular process regulated bystore-operated calcium entry. Processes regulated by store-operatedcalcium entry include, but are not limited to, calmodulin activation,calcineurin activation, mast cell degranulation and release ofinflammatory mediators, activation of calcium-dependent transcriptionfactors (e.g. nuclear factor of activated T cells (NFAT), nuclear factorkappa B (NFκB), and/or c-Jun N-terminal kinase (JNK)), NFATdephosphorylation, NFAT nuclear translocation, NFAT-dependent generegulation, expression, release and/or activity of molecules regulatedby such transcription factors (e.g. cytokine expression, release oractivity cytokine release). Exemplary calcium entry-mediated events areshown, for example, in FIG. 1.

An effect on intracellular calcium can be assessed by detecting acalcium-entry mediated event. For example, an effect on intracellularcalcium can be assessed by detecting or determining activity ofcalcium-regulated proteins, such as calmodulin and calcineurin;regulation, localization and/or activity of calcium regulatedtranscription factors such as NFAT, JNK and NFκB; and effects on geneexpression, such as genes regulated by calcium-regulated transcriptionfactors, for example, cytokine gene expression such as expression ofIL-2, IL-3, IL-4, IL-5, IL-8, IL-13, as well as tumor necrosis factoralpha (TNFα), granulocyte colony-stimulating factor (GCSF), andgamma-interferon (γ-IFN) and/or reporter genes linked to promoters orregulatory elements of such genes. An effect on intracellular calciumcan also be assessed by detecting or determining secretion and/orrelease of peptides and proteins, such as secretion of cytokines such asIL-2, and degranulation and release of inflammatory mediators such ashistamine and β-hexosaminidase.

As used herein, “modulation” with reference to intracellular calciumrefers to any alteration or adjustment in intracellular calciumincluding but not limited to alteration of calcium concentration in thecytoplasm and/or intracellular calcium storage organelles, e.g.,endoplasmic reticulum, and alteration of the amplitude or kinetics ofcalcium movements or fluxes into, out of and within cells. Modulationincludes, for example, increases, up-regulation, induction, stimulation,potentiation, relief of inhibition, reduction, inhibition,down-regulation and suppression.

As used herein, “protein involved in modulating intracellular calcium”refers to any cellular protein that is involved in regulating,controlling and/or altering intracellular calcium. For example, such aprotein can be involved in altering or adjusting intracellular calciumin a number of ways, including, but not limited to, through themaintenance of resting or basal cytoplasmic calcium levels, or throughinvolvement in a cellular response to a signal that is transmitted in acell through a mechanism that includes a deviation in intracellularcalcium from resting or basal states. In the context of a “proteininvolved in modulating intracellular calcium,” a “cellular” protein isone that is associated with a cell, such as, for example, a cytoplasmicprotein, a plasma membrane-associated protein or an intracellularmembrane protein. Proteins that modulate intracellular calcium include,but are not limited to, ion transport proteins, calcium-binding proteinsand regulatory proteins that regulate ion transport proteins. When aprotein is referred to as being “involved in” a particular aspect ofintracellular calcium or intracellular calcium regulation it can be aprotein which has the following property: when expression or activity ofthe protein in a cell is reduced, altered or eliminated, there is aconcomitant or associated alteration (including, for example, reduction,elimination, increase or other alteration) of one or more aspects ofintracellular calcium or intracellular calcium regulation. Such analteration or reduction in expression or activity can occur by virtue ofan alteration of expression of a gene encoding the protein or byaltering the levels of the protein. A protein involved in an aspect ofintracellular calcium, such as, for example, store-operated calciumentry, thus, can be one that provides for or participates in an aspectof intracellular calcium or intracellular calcium regulation. Forexample, a protein that provides for store-operated calcium entry can bean ion transport protein that forms the pore of a calcium channel and aprotein that participates in store-operated calcium entry can be aregulatory protein that modulates a store-operated calcium entrychannel. A protein that participates in store-operated calcium entry canbe a protein that is not necessarily a component of the store-operatedcalcium entry channel but is directly or indirectly associated with itsactivity, such as, for example, a non pore-forming subunit or ligand orother modulatory or regulatory protein that modulates its activity.

As used herein, a “protein that is a component of a calcium channel” isa protein that participates in a multi-protein complex that forms thechannel.

As used herein, “basal” or “resting” with reference to cytosolic calciumlevels refers to the concentration of calcium in the cytoplasm of acell, such as, for example, an unstimulated cell, that has not beensubjected to a condition that results in movement of calcium into or outof the cell or within the cell. The basal or resting cytosolic calciumlevel can be the concentration of free calcium (i.e., calcium that isnot bound to a cellular calcium-binding substance) in the cytoplasm of acell, such as, for example, an unstimulated cell, that has not beensubjected to a condition that results in movement of calcium into or outof the cell.

As used herein, “movement” with respect to ions, including cations,e.g., calcium, refers to movement or relocation, such as for exampleflux, of ions into, out of, or within a cell. Thus, movement of ions canbe, for example, movement of ions from the extracellular medium into acell, from within a cell to the extracellular medium, from within anintracellular organelle or storage site to the cytosol, from the cytosolinto an intracellular organelle or storage site, from one intracellularorganelle or storage site to another intracellular organelle or storagesite, from the extracellular medium into an intracellular organelle orstorage site, from an intracellular organelle or storage site to theextracellular medium and from one location to another within the cellcytoplasm.

As used herein, “cation entry” or “calcium entry” into a cell refers toentry of cations, such as calcium, into an intracellular location, suchas the cytoplasm of a cell or into the lumen of an intracellularorganelle or storage site. Thus, cation entry can be, for example, themovement of cations into the cell cytoplasm from the extracellularmedium or from an intracellular organelle or storage site, or themovement of cations into an intracellular organelle or storage site fromthe cytoplasm or extracellular medium. Movement of calcium into thecytoplasm from an intracellular organelle or storage site is alsoreferred to as “calcium release” from the organelle or storage site.

As used herein, “agent that modulates intracellular calcium” refers toany substance that can modulate intracellular calcium. Examples ofagents include, but are not limited to, small organic molecules, largeorganic molecules, amino acids, peptides, polypeptides, nucleotides,nucleic acids (including DNA, cDNA, RNA, antisense RNA and any double-or single-stranded forms of nucleic acids), polynucleotides,carbohydrates, lipids, lipoproteins, glycoproteins, inorganic ions(including, for example, Gd³⁺, lead and lanthinum).

As used herein, a “STIM protein” includes any STIM protein, includingbut not limited to, STIM1, a STIM, DSTIM, CSTIM and STIM-like proteins.STIM1, STIM2, DSTIM and STIM-like protein are described in detail below.A STIM-like protein can be one that has similar homology to STIM1 andSTIM2 proteins and that is at least about 45% homologous to the proteinencoded by Drosophila gene CG9126 over at least about 52% of theprotein.

As used herein, a “biological activity of a STIM1 protein” includes anyactivity known to those of skill in the art to be associated with STIM1,and also the intracellular calcium modulating activity described herein.STIM1 biological activities known to those of skill in the art, include,but are not limited to, binding to pre-B cells and differentiated Blymphocytes, augmentation of IL-7-dependent proliferation of pre-Bcells, modulation of cell morphology and suppression of tumor growth.

As used herein, “mammalian STIM1 proteins” refer collectively tomammalian STIM1 proteins such as those as set forth and/or encoded bySEQ ID NOs: 3, 4, 9, 10, 49-56, 85, 95, 96, 97, 98 and proteins with atleast 90% homology over at least 70% of the protein with human STIM1and/or SEQ ID NO: 4. Mammalian STIM1 proteins include naturallyoccurring variants of STIM1 proteins found in mammals as well asvariants constructed synthetically or recombinantly that retain at least90% homology over at least 70% of the protein with human STIM1 and/orSEQ ID NO: 4.

As used herein, “reference STIM1” refers to nucleotide sequence as setforth in SEQ ID NO: 52 encoding reference STIM1 protein set forth in SEQID NO: 52. Reference STIM1 contains a hamster partial STIM1 cDNA thatwas extended with 5′ and 3′ sequences from the rat Rattus norvegicuschromosome 1 WGS supercontig sequence having GenBank Accession No.NW_(—)043388.

As used herein, “rodent reference STIM1” refers collectively to rat,hamster and reference STIM1. Rodent reference STIM1 nucleotide sequencesrefers collectively to rat, hamster and reference STIM1 nucleotidesequences and include SEQ ID NOs: 51, 95 and 97. Rodent reference STIM1proteins or amino acid sequences refer collectively to rat, hamster andreference STIM1 proteins or amino acid sequences, and include SEQ IDNOs: 52, 96 and 98 and proteins as encoded by SEQ ID NOs: 51, 95 and 97.

As used herein, “overexpression” refers to the expression of a proteinin a test cell such that it is greater than the expression of theprotein in a reference cell. The reference cell can be a cell that issubstantially the same but lacks the means for overexpressing theprotein. For example, the test cell can contain one or more heterologousnucleic acid molecules encoding a protein not present or not expressedin the reference cell. The test cell can be induced by a agent orcondition resulting in expression of a protein and the reference cellcan be an uninduced cell. The reference cell and test cell are notrequired to be assessed simultaneously. They can be assessed separately,under substantially the same conditions. The reference cell can also berepresented by a collection of data, for example stored in a database;the data can be collected from experiments of cells and conditions fromone or more experiments which can be used for comparison with a testcell.

As used herein, “test agent,” in the context of methods for identifyingagents that modulate intracellular calcium, refers to any substance thatis being evaluated as a possible agent that modulates intracellularcalcium.

As used herein, “agent that modulates the level and/or activity of aprotein” refers to any substance that can modulate the amount of and/oractivity of a protein. Such agents include, but are not limited to,small and large organic molecules, amino acids, peptides, polypeptides,nucleotides, nucleic acids (including DNA, cDNA, expression vectors,RNA, antisense RNA, and any double- or single-stranded forms of nucleicacids), polynucleotides, carbohydrates, lipids, lipoproteins andglycoproteins.

As used herein, “amelioration” refers to an improvement in a disease orcondition or at least a partial relief of symptoms associated with adisease or condition.

As used herein, “cell response” refers to any cellular response thatresults from ion movement into or out of a cell or within a cell. Thecell response may be associated with any cellular activity that isdependent, at least in part, on ions such as, for example, calcium. Suchactivities may include, for example, cellular activation, geneexpression, endocytosis, exocytosis, cellular trafficking and apoptoticcell death.

As used herein, “nucleic acid” or “nucleic acid molecule” refers to bothdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, aswell as modified polynucleotides. Modified polynucleotides includepolynucleotides having, for example, modifications to the base, sugar,or the phosphate portion, or that contain a modified phosphodiesterlinkage. The term “nucleic acid” includes both single-stranded anddouble-stranded nucleic acids, which can correspond to the sense strand,antisense strand or both of a reference sequence. The term includesmolecules with various conformations, including linear, circular,hairpin and branched molecules.

As used herein, “heterologous” or “foreign” with reference to nucleicacids, cDNA, DNA and RNA are used interchangeably and refer to nucleicacid, DNA or RNA that does not occur naturally as part of the genome inwhich it is present or which is found in a location(s) or in an amountin the genome that differs from that in which it occurs in nature. Itcan be nucleic acid that has been exogenously introduced into the cell.Thus, heterologous nucleic acid is nucleic acid not normally found inthe host genome in an identical context. Examples of heterologousnucleic acids include, but are not limited to, DNA that encodes a geneproduct or gene product(s) of interest, introduced, for example, forpurposes of gene therapy or for production of an encoded protein. Otherexamples of heterologous DNA include, but are not limited to, DNA thatencodes a selectable marker, DNA that encodes therapeutically effectivesubstances, such as enzymes and hormones, and DNA that encodes othertypes of proteins, such as antibodies.

As used herein, “expression” refers to the process by which nucleicacid, e.g., DNA, is transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the nucleic acid is derived from genomicDNA, expression may, if an appropriate eukaryotic host cell or organismis selected, include splicing of the mRNA.

As used herein, “vector” or “plasmid” refers to discrete elements thatare used to introduce heterologous nucleic acids into cells. Typically,vectors are used to transfer heterologous nucleic acids into cells foreither expression of the heterologous nucleic acid or for replication ofthe heterologous nucleic acid. Selection and use of such vectors andplasmids are well within the level of skill of the art.

As used herein, “transformation” or “transfection” refers to the processby which nucleic acids are introduced into cells. Transfection refers tothe taking up of exogenous nucleic acid, e.g., an expression vector, bya host cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan. Successful transfection is generally recognized by detection ofthe presence of the heterologous nucleic acid within the transfectedcell, such as, for example, any visualization of the heterologousnucleic acid or any indication of the operation of a vector within thehost cell.

As used herein, the “amino acids,” which occur in the various amino acidsequences appearing herein, are identified according to theirwell-known, three-letter or one-letter abbreviations (see Table 1). Thenucleotides, which occur in the various DNA fragments, are designatedwith the standard single-letter designations used routinely in the art.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion hydrolysis) of a polypeptide at its peptide linkages.The amino acid residues described herein are preferably in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide. NH2 refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxyl terminus of apolypeptide. In keeping with standard polypeptide nomenclature describedin J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. §§.1.821-1.822, abbreviations for amino acid residues are shown in Table 1:TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence and modified and unusualamino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, andincorporated herein by reference. Furthermore, it should be noted that adash at the beginning or end of an amino acid residue sequence indicatesa peptide bond to a further sequence of one or more amino acid residuesor to an amino-terminal group such as NH₂ or to a carboxyl-terminalgroup such as COOH.

In a peptide or protein, suitable conservative substitutions of aminoacids are known to those of skill in this art and may be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224).

Such substitutions may be made in accordance with those set forth inTABLE 2 as follows: TABLE 2 Original Conservative residue substitutionAla (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) AsnGlu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L)Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu;Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile;LeuOther substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the amino acid sequences of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences andthe residues contained therein. Methods for assessing the degree ofsimilarity between proteins or nucleic acids are known to those of skillin the art. For example, in one method of assessing sequence similarity,two amino acid or nucleotide sequences are aligned in a manner thatyields a maximal level of identity between the sequences. “Identity”refers to the extent to which the amino acid or nucleotide sequences areinvariant. Alignment of amino acid sequences, and to some extentnucleotide sequences, also can take into account conservativedifferences and/or frequent substitutions in amino acids (ornucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g.: Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exist a number of methodsto measure identity between two polynucleotide or polypeptide sequences,the term “identity” is well known to skilled artisans (Carillo, H. &Lipton, D., SIAM J Applied Math 48:1073 (1988)).

As used herein, “homology” with reference to proteins or nucleic acidsrefers to shared sequence similarity that takes into account bothidentical residues and residues that may substitute for one another.Substitutions may include, for example, conserved amino acids andfrequent substitutions based on statistical analyses and evolutionarydistance.

Percent identity and percent homology may be determined, for example, bycomparing sequence information using any of a number of computeralgorithms known in the art. The GAP program uses the alignment methodof Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), as revised bySmith and Waterman (Adv. Appl. Math. 2:482 (1981)). Briefly, the GAPprogram defines similarity as the number of aligned symbols (i.e.,nucleotides or amino acids) which are similar, divided by the totalnumber of symbols in the shorter of the two sequences. Defaultparameters for the GAP program may include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix of Gribskov and Burgess, Nucl. AcidsRes. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLASOF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical ResearchFoundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and anadditional 0.10 penalty for each symbol in each gap; and (3) no penaltyfor end gaps.

Whether any two protein or nucleic acid molecules have amino acid ornucleotide sequences that are at least, for example, 20%, 30%, 40%, or50%, “identical” can be determined using known computer algorithms suchas the “FAST A” program, using for example, the default parameters as inPearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988). The BLASTfunction of the National Center for Biotechnology Information databasemay be used to determine identity. By way of example, in a comparison ofa test and reference polypeptide, wherein the length of the referencepolypeptide is 100 amino acids, a level of 90% or more identity betweenthe polypeptides is indicative of having no more than 10% (i.e., 10 outof 100) amino acids in the test polypeptide differing from that of thereference polypeptide. Similar comparisons may be made between a testand reference polynucleotides. Such differences may be represented aspoint mutations randomly distributed over the entire length of an aminoacid sequence or they may be clustered in one or more locations ofvarying length up to the maximum allowable, e.g. 10/100 amino aciddifference (approximately 90% identity). The BLinK tool (“BLAST Link”)displays the results of precomputed BLAST searches (Altschul et al.(19990) J. Mol. Biol. 215:403-410) that have been done for proteinsequences in the Entrez Proteins data domain against the non-redundant(nr) database (e.g., GenBank).

Methods commonly employed to determine identity or homology between twosequences include, but are not limited to, those disclosed in Guide toHuge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carillog, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).Methods to determine identity and homology are codified in computerprograms. Computer program methods to determine identity and homologybetween two sequences include, but are not limited to, GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)),BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403(1990)).

Therefore, as used herein, the terms “identity” and “homology” representa comparison between two polypeptides or polynucleotides. Typically, a“test” polypeptide or polynucleotide is compared to a “reference”molecule to determine if there is a significant level of similarity(including identity and/or homology) between the test and referencemolecules.

As used herein, the terms at least “X % homology over X % of theprotein” or “X % identity over X % of the protein” with reference to acomparison of protein A with protein B refers to protein A having X %homology or X % identity to protein B over X % of the amino acidsequence of protein B. The term “over the protein” means over the lengthof the amino acid sequence of the protein, but not necessarily over acontiguous sequence of amino acids of the protein. Thus, for example,protein B may be 40% homologous to protein A over 60% of protein B;however, the 60% of the amino acids in protein B to which protein A hashomology may be located in multiple, separate sequences (e.g., regionsor domains) of protein B.

As used herein, protein “homologs” refers to similar proteins encoded byrelated but different genes either within a species or between species.Protein “orthologs” refers to similar proteins in different species thatarose from a common ancestral gene.

As used herein, all assays and procedures, such as hybridizationreactions and antibody-antigen reactions, unless otherwise specified,are conducted under conditions recognized by those of skill in the artas standard conditions.

As used herein, a “nucleotide sequence” or “sequence of nucleotides” (orreference to a nucleic acid molecule designated by a SEQ ID NO:) refersto a single-stranded nucleic acid molecule having the referencesequence, to its reverse complement, or to a double-stranded orpartially double-stranded molecule containing the reference sequencebase-paired to its reverse complement. Accordingly, a nucleic acidmolecule that hybridizes to a reference nucleotide sequence canhybridize to the reference sequence or to its reverse complement.

As used herein, “isolated,” with reference to an invention biomolecule,such as a nucleic acid molecule, oligonucleotide, -polypeptide orantibody, indicates that the molecule has been altered by the hand ofman from how it is found in its natural environment. For example, amolecule produced by and/or contained within a recombinant host cell isconsidered “isolated.” Likewise, a molecule that has been purified,partially or substantially, from a native source or recombinant hostcell, or produced by synthetic methods, is considered “isolated.”Depending on the intended application, an isolated molecule can bepresent in any form, such as in an animal, cell or extract thereof;dehydrated, in vapor, solution or suspension; or immobilized on a solidsupport.

As used herein, an “amino acid substitution relative to” a referencesequence, refers to a difference between the amino acid residue in thereference sequence and the amino acid residue at the “correspondingposition” in the second sequence. Corresponding positions can bedetermined by comparing and aligning sequences to maximize the number ofidentical residues. The position of interest is then given the positionnumber assigned to the aligned residue in the reference sequence. Anamino acid substitution can either be conservative or non-conservative.Conservative amino acid substitutions include, but are not limited to,substitution of an apolar amino acid with another apolar amino acid(such as replacement of leucine with an isoleucine, valine, alanine,proline, tryptophan, phenylalanine or methionine); substitution of acharged amino acid with a similarly charged amino acid (such asreplacement of a glutamic acid with an aspartic acid, or replacement ofan arginine with a lysine or histidine); substitution of an unchargedpolar amino acid with another uncharged polar amino acid (such asreplacement of a serine with a glycine, threonine, tyrosine, cysteine,asparagine or glutamine); or substitution of a residue with a differentfunctional group with a residue of similar size and shape (such asreplacement of a serine with an alanine; an arginine with a methionine;or a tyrosine with a phenylalanine).

As used herein, “position,” with regard to a specified nucleotide oramino acid, refers to the numerical location of the nucleotide or aminoacid in the reference sequence, with the 5′-most nucleotide, or theN-terminal amino acid, designated as position 1.

As used herein, “operatively linked” indicates that the recitednucleotide sequences are positioned such that there is a functionalrelationship between the sequences. In the context of RNA transcription,the term “operatively linked” indicates that a regulatory sequence(s),such as a promoter, is positioned in such a manner to permit or modulatetranscription of RNA using the linked sequence as a template, whenappropriate molecules of the transcriptional machinery are bound to theregulatory sequence(s). Two sequences that are “operatively linked” arenot necessarily contiguous. Methods for operatively linking a nucleicacid to a promoter are well known in the art and include, for example,cloning the nucleic acid into a vector containing the desired promoter,or appending the promoter to a nucleic acid sequence using PCR.

As used herein, “promoter of gene expression” refers to those nucleotidesequences containing binding sites for RNA polymerase and othertranscription factors necessary for transcription. The choice ofpromoter to operatively link to an invention nucleic acid molecule willdepend on the intended application, and can be determined by thoseskilled in the art. For example, it may be desirable to link theinvention nucleic acid molecule to a regulated promoter, such that geneexpression can be turned on or off. Alternatively, it may be preferredto have expression driven by either a weak or strong constitutivepromoter. Exemplary promoters suitable for bacterial and in vitrotranscription systems include, for example, T7, T3 and SP6 promoters.Promoters suitable for mammalian cell systems include, for example, SV40early promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV)promoter, adenovirus major late promoter, and Moloney murine leukemiavirus (MMLV) promoter. Regulated promoters can include inducible andrepressible elements, such as ecdysone-responsive elements,steroid-responsive elements, lac or trp inducible elements, and thelike.

As used herein, “oligonucleotide” refers to a short nucleic acidmolecule, such as a nucleic acid molecule that contains, or consists of,at least about 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 35, 40, 45,50 or more nucleotides. An oligonucleotide can optionally contain, orconsist of, no more than about 500, 400, 350, 300, 250, 200, 150, 100,75, 50, 45, 40, 35, 30 or 25 nucleotides. An oligonucleotide can beentirely single-stranded or double-stranded, or partiallysingle-stranded and partially double-stranded, as appropriate for theintended application.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated that agiven nucleic acid molecule can serve as both a “probe” and as a“primer.” A primer can be used in a variety of methods, includingpolymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNAPCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′and 5′ RACE, in situ PCR, and ligation-mediated PCR.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the 5′ end of a sequence to beamplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizeswith the complement of the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. aninvention oligonucleotide) to a target nucleic acid molecule. Those ofskill in the art are familiar with both in vitro and in vivo parametersthat affect specific hybridization, such as length and composition ofthe particular molecule. Parameters particularly relevant to in vitrohybridization further include annealing and washing temperature, buffercomposition and salt concentration. Exemplary washing conditions forremoving non-specifically bound nucleic acid molecules at highstringency are 0.1× SSPE, 0.1% SDS, 65° C., and at medium stringency are0.2× SSPE, 0.1% SDS, 50° C. Equivalent stringency conditions are knownin the art. The skilled person can readily adjust these parameters toachieve specific hybridization of a nucleic acid molecule to a targetnucleic acid molecule appropriate for a particular application.

As used herein, “inhibitory oligonucleotide” refers to anoligonucleotide that specifically hybridizes to a target nucleic acidmolecule in a cell, thereby specifically reduces the amount of targetmRNA or its encoded protein present in the cell. Inhibitoryoligonucleotides can be used, for example, in functional genomicsapproaches to determine the biological activities of a target gene, orto treat conditions associated with expression of the target mRNA.Examples of inhibitory oligonucleotides include, but are not limited to,interference nucleic acid molecules, antisense nucleic acid moleculesand catalytic nucleic acid molecules. As with other oligonucleotidesdescribed herein, inhibitory oligonucleotides can be producedsynthetically, or can be produced recombinantly by either in vitro or invivo transcription, by methods known in the art. Methods of usinginhibitory oligonucleotides in ex vivo and in vivo applications are alsowell known in the art, and such methods can be used to reduce the amountof target mRNA or protein.

As used herein, “interference nucleic acid molecule” refers to amolecule that acts through a complex known as the RNA-induced silencingcomplex (RISC) to specifically induce the degradation of cognate mRNA.Interference nucleic acid molecules may additionally or alternativelyact to inhibit mRNA translation through the same or a different complex.Interference nucleic acid molecules include, but are not limited to,long double-stranded RNAs (dsRNA) that can be cleaved intracellularlyinto short inhibitory RNAs (siRNA); siRNAs, which are about 21-23nucleotide double-stranded RNA molecules, generally with 2 nucleotidesingle stranded overhangs (Hannon (2002) Nature 418:244-251); shorthairpin RNAs (shRNA), which are similar to small temporal RNAs (stRNAs)and micro-RNAs (mRNAs) (McManus et al. (2002) RNA 8:842-850); andmRNA-cDNA hybrids (D-RNAi; Lin et al. (2001) Biochem. Biophys. Res.Commun. 281:639-644). Interference molecules can thus be completely orpartially double-stranded.

As used herein, “antisense nucleic acid molecule” refers to a moleculethat base pairs with cognate mRNA and inhibit its translation. Theantisense-bound mRNA is then degraded by RnaseH, or alternatively formsa stable complex that blocks a cell's translational machinery. Antisensemolecules can be DNA or RNA, and commonly contain modified bases, sugarsor linkages to enhance their stability. Methods of making antisensenucleic acids by synthetic and recombinant methods are known in the art(e.g. Phillips (ed.) (1999) “Antisense Technology, Part A (Methods inEnzymology, Volume 313)” Academic Press).

As used herein, “catalytic nucleic acid molecule” refers a molecule thatbase-pairs with, and enzymatically cleaves, target RNA. Catalyticnucleic acid molecules include, for example, hairpin ribozymes,hammerhead ribozymes, hepatitis delta virus ribozymes, andlead-dependent ribozymes (Doherty and Doudna (2001) Annu. Rev. Biophys.Biomol. Struct. 30:457-475). Methods of making catalytic nucleic acidmolecules by synthetic and recombinant methods are known in the art(e.g. Gibson (ed.) (2002) “Antisense and Ribozyme Methodology:Laboratory Companion” John Wiley & Sons).

As used herein, “domain” refers to a region of a polypeptide that iscapable of maintaining its structure or function in a context other thanin the full-length polypeptide, such as when expressed alone or as afusion protein. A structural domain can comprise one or more betasheets, alpha helices, loops, folds or other structural motifs, and cancorrespond, for example, to one or more extracellular domains,transmembrane domains or intracellular domains. A functional domain can,but need not, correspond exactly to a structural domain. Exemplaryfunctional domains include an epitope, a ligand or effector bindingsite, a modification site (e.g. a site of phosphorylation, acylation orglycosylation), an enzyme active site, and the like. Regions of apolypeptide that comprise structural or functional domains can bedetermined using methods known in the art. For example, the boundariesof a single exon, or of several contiguous exons, are often identical tothe boundaries of a structural or functional domain. Domains can also beidentified by their homology to proteins known to contain particularstructural or functional domains, for example, by using the search andalignment resources of the Pfam database (http://pfam.wustl.edu/;Bateman et al. (2002) Nucleic Acids Research 30:276-280). Domains canalso be identified by producing polypeptide fragments and directlyanalyzing their structural or functional properties.

As used herein, “mature” with respect to a STIM1 polypeptide, refers tothe polypeptide with the presumptive signal peptide (amino acids 1-22 ofhuman, mouse or reference STIM1) removed.

As used herein, “extracellular domain of STIM1” refers to the portion ofthe mature polypeptide that is N-terminal to the presumptivetransmembrane domain (e.g. N-terminal to the position corresponding toposition 214 of human, mouse or reference STIM1). It will be appreciatedthat the “extracellular domain” may not necessarily be localizedextracellularly in all instances, and thus may alternatively belocalized intralumenally or intracytoplasmically (e.g. during proteinsynthesis, sorting, or degradation, or when synthesized as a recombinantfusion protein).

As used herein, “cytoplasmic domain of STIM1” refers to the portion ofthe mature polypeptide that is C-terminal to the presumptivetransmembrane domain (e.g. C-terminal to the position corresponding toposition 234 of human, mouse or reference STIM1). It will be appreciatedthat the “cytoplasmic domain” may not necessarily be localized to thecytoplasm in all instances, and thus may alternatively be localizedintralumenally or extracellularly (e.g. during protein synthesis,sorting, or degradation, or when synthesized as a recombinant fusionprotein).

As used herein, “polypeptide” includes naturally and non-naturallyoccurring peptides and proteins. A polypeptide can be a core protein, orcan be a post-translationally modified form of a protein such as aphosphoprotein, glycoprotein, proteoglycan, lipoprotein ornucleoprotein. Polypeptides may include one or more D-amino acids, oneor more chemically or enzymatically derivatized amino acids (e.g.derivatized by replacement of a hydrogen by an alkyl, acyl, or aminogroup; esterification of a carboxyl group with a suitable alkyl or arylmoiety; alkylation of a hydroxyl group to form an ether derivative;phosphorylation or dephosphorylation of a serine, threonine or tyrosineresidue; or N- or O-linked glycosylation), one or more amino acidanalogs or mimetics (described, for example, in Sawyer, “Peptide BasedDrug Design,” ACS, Washington (1995)), or one or more labeled (e.g.radiolabeled or fluorescently labeled) amino acids.

As used herein, “peptide” refers to a short polypeptide, such as apolypeptide that contains, or consists of, at least about 8, 9, 10, 12,14, 16, 18, 20, 25, 30, 35, 40 or more amino acids. A peptide canoptionally contain, or consist of, no more than about 200, 150, 100, 75,50, 45, 40, 35, 30, 25 or 20 amino acids.

As used herein, “antibody” is intended to refer to both polyclonal andmonoclonal antibodies, as well as molecules containing one or moreantigen binding fragments of such antibodies (e.g. complementaritydetermining regions (CDRs), Fab, F(ab′)₂, Fd and Fv fragments and thelike). In addition, the term “antibody” is intended to encompassnon-natural antibodies, including, for example, single chain antibodies,chimeric antibodies, bifunctional antibodies, humanized antibodies,CDR-grafted antibodies and CDR-grafted alternative scaffolds, as well asantigen-binding fragments thereof. Also encompassed are detectablylabeled antibodies, such as antibodies labeled with a radioactiveisotope, fluorochrome, ferromagnetic substance, luminescent agent,enzyme or ligand.

As used herein, “specifically binds,” in the context of anantibody-antigen interaction, refers to a binding interaction betweenthe antibody and the target antigen that has a K_(d) of about 10⁻⁶ M orless, such as about 10⁻⁷ M or less, including about 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or less. In contrast, the term “does not bind,” in the contextof an antibody-antigen interaction, refers to a binding interactionbetween the antibody and the target antigen that has a K_(d) of about10⁻⁵ M or higher. Methods of determining binding affinity betweenantibodies and antigens are well known in the art (see, for example,Harlow and Lane (1989) “Antibodies: A Laboratory Manual,” Cold SpringHarbor Press (New York)).

As used herein, “kit” refers to a product that includes at least onecontainer having disposed therein an amount of an invention molecule(e.g. nucleic acid molecule, polypeptide, peptide, antibody, agent,etc.) sufficient for an intended use of the kit. The container can be ofany convenient material and shape, including those customarily used inlaboratories and clinics, and can serve to provide a contaminant-free orsterile environment for the contained molecule. A kit can furtherinclude a tangible description of the amount or concentration of themolecule, and/or guidance regarding its formulation and use. A kit canfurther include additional containers, such as containers havingdisposed therein buffers, carriers, solvents and the like, useful forformulating the molecule, and/or containers having disposed thereinadditional molecules used in conjunction with the molecules providedherein.

As used herein, “substrate” refers to any suitable solid or semi-solidsupport, such as membranes, filters, chips, slides, wafers, fibers,magnetic or nonmagnetic beads, gels, tubing, plates, polymers,microparticles and capillaries. The substrate can have a variety ofsurface forms, such as wells, trenches, pins, channels and pores, towhich an invention molecule (e.g. nucleic acid molecule, polypeptide,peptide, antibody, agent, etc.) can be bound.

As used herein, “immune cells” include cells of the immune system andcells that perform a function or activity in an immune response, suchas, but not limited to, T-cells, B-cells, lymphocytes, macrophages,dendritic cells, neutrophils, eosinophils, basophils, mast cells, plasmacells, white blood cells, antigen presenting cells and natural killercells.

As used herein, “cytokine” refers to small soluble proteins secreted bycells that can alter the behavior or properties of the secreting cell oranother cell. Cytokines bind to cytokine receptors and trigger abehavior or property within the cell, for example, cell proliferation,death or differentiation. Exemplary cytokines include, but are notlimited to, interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18,IL-1α, IL-1β, and IL-1 RA), granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),oncostatin M, erythropoietin, leukemia inhibitory factor (LIF),interferons, B7.1 (also known as CD80), B7.2 (also known as B70, CD86),TNF family members (TNF-α, TNF-β, LT-β, CD40 ligand, Fas ligand, CD27ligand, CD30 ligand, 4-1BBL, Trail), and MIF.

As used herein, “a system” is used interchangeably with the term“combination” and refers to any association between or among two or moreitems. The combination can be two or more separate items, can be amixture thereof or any variation thereof.

B. Cellular Signaling and Calcium Modulation

All living cells sense and respond to their environment by a set ofmechanisms termed cell signaling. These mechanisms are part of a complexsystem of communication that governs basic cellular activities andcoordinates the actions of cells. Living cells must respondappropriately to their environment, whether they are free-living in thesoil or part of a tissue. Cell communication is necessary for theexistence of multicellular organisms. The ability of cells to perceiveand correctly respond to their microenvironment is the basis ofdevelopment, tissue repair, and immunity as well as normal tissuehomeostasis.

Cells, through a class of proteins known as receptors, receiveinformation from their environment. The information is then processedthrough signaling pathways and decoded in the nucleus and other areas ofthe cell. The spatial and temporal dynamics of both receptors and thecomponents of the signaling pathways are important in the transfer ofthe message from the extracellular to the intracellular environment. Thecomponents of the signaling pathway, their location either inside or onthe surface of the cell, the role of each component in the transductionof the signal, and interactions among the components of the system inorder to transduce the signal are required to elucidate the cause,mechanism and effect of the signal transduction on the cell. Moleculesthat can serve as components of a signaling pathway include, but are notlimited to, an organic compound, inorganic compound, metal complex,receptor, enzyme, antibody, protein, nucleic acid, peptide nucleic acid,DNA, RNA, polynucleotide, oligonucleotide, oligosaccharide, lipid,lipoprotein, amino acid, peptide, polypeptide, peptidomimetic,carbohydrate, cofactor, drug, prodrug, lectin, sugar, glycoprotein,hormone, steroid, biomolecule, macromolecule, biopolymer, polymer,sub-cellular structure, sub-cellular compartment or any combination,portion, salt, or derivative thereof, a virus, such as a viral vector orviral capsid with or without packaged nucleic acid, phage, including aphage vector or phage capsid, with or without encapsulated nucleic acid,a cell, including eukaryotic and prokaryotic cells or fragments thereof,a liposome or micellar agent or other packaging particle, and other suchbiological materials. The interactions among the components of thesignaling system include, but are not limited to, protein:protein,protein:nucleic acid, nucleic acid:nucleic acid, protein:lipid,lipid:lipid, protein:small molecule, receptor:signal, antibody:antigen,peptide nucleic acid:nucleic acid, and small molecule:nucleic acid.

There are many signaling pathways; such pathways include, but are notlimited to, the Notch pathway; G-protein signaling and activation ofphospholipase C (PLC); nuclear factor κB (NFκB) pathway; T cellactivation; activation of protein kinase C (PKC) and protein kinase A(PKA); Ahr signal transduction pathway; anthrax toxin mechanism ofaction; ataxia telangiectasia-mutated gene (ATM) signaling pathway;bioactive peptide induced signaling pathway; co-activator-associatedarginine methyltransferase 1 (CARM1) and regulation of the estrogenreceptor; CBL mediated ligand-induced down-regulation of EGF receptors;control of skeletal myogenesis by HDAC and calcium/calmodulin-dependentkinase; CXCR4 signaling pathway; cyclins and cell cycle regulation; thecytokine network; D4-GDI (GDP dissociation inhibitor) signaling pathway;mitogen activated protein kinase (MAPK) pathway; Erk1/Erk2 MAPKsignaling pathway; FAS signaling pathway; Fc Epsilon Receptor Isignaling; growth hormone signaling pathway; ErbB4 signaling; interferonsignaling, such as, interferon alpha (IFNα) and IFNγ signaling;insulin-like growth factor 1 (IFG-1) signaling; interleukin (IL)signaling, such as, IL-17, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6 and IL-22signaling; insulin signaling pathway; integrin signaling pathway;phorbal esters signaling pathway; tumor necrosis factor receptor 5(TMFR-5) pathway; p53 signaling pathway; CCR5 signaling pathway;phospholipase C and phospholipase C epsilon pathways; RAC1 cell motilitysignaling pathway; RAS signaling pathway; reelin signaling pathway; RHOcell motility signaling pathway; ubiquitin-proteasomal pathway; signaltransducers and activators of transcription 3 (STAT-3) pathway;transforming growth factor β (TFGβ) signaling pathway; TNF/Stressrelated signaling pathways; TNFR1 and TNFR2 signaling pathways; TRKAreceptor signaling pathway; lymphocyte signaling; Atk signaling; andchromatin regulation.

Errors in cellular information processing contribute to numerousdiseases, including, but not limited to, cancer, autoimmunity, anddiabetes. In order to effectively treat these diseases, knowledge of themechanism by which the signal is transduced is required. Identifying thecomponents of the signaling pathway and the message that it transmitsallows for the alteration, blocking or amplification of the message andpossible prevention of the undesired cellular response or activation ofdesired cellular response. This information can also be used in otherways such as, but not limited to, controlling the behavior of individualcells and permitting the creation of artificial tissues.

A key component in some signaling pathways is calcium. Intracellularcalcium concentration is tightly regulated, and numerous cellularconstituents, e.g., proteins, and processes are calcium sensitive. Thus,many signal transduction mechanisms involve transient calcium fluxacross the plasma membrane and membranes of intracellular organelles.

1. Calcium Homeostasis

Cellular calcium homeostasis is a result of the summation of regulatorysystems involved in the control of intracellular calcium levels andmovements. Cellular calcium homeostasis is achieved, at least in part,by calcium binding and by movement of calcium into and out of the cellacross the plasma membrane and within the cell by movement of calciumacross membranes of intracellular organelles including, for example, theendoplasmic reticulum, sarcoplasmic reticulum, mitochondria andendocytic organelles including endosomes and lysosomes.

Movement of calcium across cellular membranes is carried out byspecialized proteins. For example, calcium from the extracellular spacecan enter the cell through various calcium channels and a sodium/calciumexchanger and is actively extruded from the cell by calcium pumps andsodium/calcium exchangers. Calcium can also be released from internalstores through inositol trisphosphate or ryanodine receptors and can betaken up by these organelles by means of calcium pumps.

Endocytosis provides another process by which cells can take up calciumfrom the extracellular medium through endosomes. In addition, somecells, e.g., exocrine cells, can release calcium via exocytosis.

a. Basal or Resting Cytosolic Calcium Levels

Cytosolic calcium concentration is tightly regulated with resting levelsusually estimated at approximately 0.1 μM in mammalian cells, whereasthe extracellular calcium concentration is typically about 2 mM. Thistight regulation facilitates transduction of signals into and withincells through transient calcium flux across the plasma membrane andmembranes of intracellular organelles. There is a multiplicity ofintracellular calcium transport and buffer systems in cells that serveto shape intracellular calcium signals and maintain the low restingcytoplasmic calcium concentration. In cells at rest, the principalcomponents involved in maintaining basal calcium levels are calciumpumps and leaks in the endoplasmic reticulum and plasma membrane.Disturbance of resting cytosolic calcium levels can effect transmissionof such signals and give rise to defects in a number of cellularprocesses. For example, cell proliferation involves a prolonged calciumsignaling sequence. Other cellular processes, including but not limitedto, secretion, fertilization and learning, involve calcium signaling.

b. Store-Operated Calcium Entry

One mechanism for movement of calcium into cells through the plasmamembrane is commonly referred to as store-operated calcium entry.Reduced calcium concentration in intracellular calcium stores such asthe endoplasmic reticulum resulting from release of calcium therefromprovides a signal for influx of calcium from the extracellular mediuminto the cell. This influx of calcium, which produces a sustained“plateau” elevation of cytosolic calcium concentration, generally doesnot rely on voltage-gated plasma membrane channels and does not involveactivation of calcium channels by calcium. This calcium influx mechanismhas been referred to as capacitative calcium entry (CCE), calciumrelease-activated, store-operated or depletion-operated calcium entry.Store-operated calcium entry can be recorded as an ionic current withdistinctive properties. In some instances, this current is referred toas I_(SOC) (store-operated current) or I_(CRAC) (calciumrelease-activated current).

i. Regulation of Store-Operated Calcium Entry by Intracellular CalciumStores

Store-operated calcium entry is regulated by the level of calcium withinan intracellular calcium store. Intracellular calcium stores can becharacterized by sensitivity to agents, which can be physiological orpharmacological, that activate release of calcium from the stores orinhibit uptake of calcium into the stores. Different cells have beenstudied in characterization of intracellular calcium stores, and storeshave been characterized as sensitive to various agents, including, butnot limited to, IP₃ and compounds that effect the IP₃ receptor,thapsigargin, ionomycin and/or cyclic ADP-ribose (cADPR) (see, e.g.,Berridge (1993) Nature 361:315-325; Churchill and Louis (1999) Am. J.Physiol. 276:C426-C434; Dargie et al. (1990) Cell Regul. 1:279-290;Gerasimenko et al. (1996) Cell 84:473-480; Gromoda et al. (1995) FEBSLett. 360:303-306; Guse et al. (1999) Nature 398:70-73).

The identities and/or cellular locations of calcium stores can bedetermined, for example, by isolation and characterization of organellesor imaging of cells using calcium-sensitive indicators which localize instorage organelles. Mag-fura 2, an example of one such indicator, is aUV light-excitable, ratiometric, low-affinity fluorescent calciumindicator. The moderate calcium affinity of mag-fura-2 and the tendencyof its acetoxymethyl (AM) ester to accumulate in subcellularcompartments makes this indicator particularly useful in monitoring ofor assessing calcium stores (see, e.g., Hofer and Machen (1993) Proc.Natl. Acad. Sci. U.S.A. 90:2598-2602; Hofer et al. (1998) EMBO J.17:1986-1995; Hofer et al. (1998) J. Cell Biol. 140:325-334; Churchilland Louis (1999) Am. J. Physiol. 276:C426-C434). Intracellular calciumstores include the endoplasmic reticulum and sarcoplasmic reticulum,which are sensitive to IP₃ or caffeine/ryanodine andthapsigargin/cyclopiazonic acid (CPA) (see, e.g., Pozzan et al. (1994)Physiol. Rev. 74:595-637; Meldolesi and Pozzan (1998) J. Cell Biol.142:1395-1398; Meldolesi and Pozzan (1998) Trends Biochem. Sci.23:10-14; Golovina and Blaustein (2000) Glia 31:15-28), and isolatedzymogen granules and the envelope of isolated nuclei, which aresensitive to cADPR and IP₃ (see, e.g., Gerasimenko et al. (1996) Cell84:473-480; Gerasimenko et al. (1995) Cell 80:439-444).

Basal free calcium concentrations in calcium stores can be orders ofmagnitude, e.g., 10³-fold, greater than the free calcium concentrationin the cytosol. For example, the basal free calcium concentrationmeasured in the endoplasmic reticulum of HEK293 cells ranges betweenabout 200-700 μM, with an average of about 500 μM, whereas the basalfree calcium concentration in the cytosol is about 50 nM (Yu and Hinkle(2000) J. Biol. Chem. 275:23648-23653).

Free calcium concentrations in the endoplasmic reticulum can be measuredin a variety of ways such as, for example, using variouscalcium-sensitive indicators (see, e.g., Yu and Hinkle (2000) J. Biol.Chem. 275:23648-23653) including mag-fura 2 (see, e.g., Hofer and Schulz(1996) Cell Calcium 20:235-242), endoplasmic reticulum-targeted aequorin(see, e.g., Montero et al. (1995) EMBO J. 14:5467-5475) and endoplasmicreticulum-targeted “cameleons” (i.e., fluorescent calcium indicatorsbased on fluorescence resonance energy transfer between two modifiedgreen fluorescent proteins (GFPs) contained in a protein with calmodulinand a calmodulin-binding peptide; see, e.g., Miyawaki et al. (1997)Nature 388:882-887 and Yu and Hinkle (2000) J. Biol. Chem.275:23648-23653).

Accumulation of calcium within endoplasmic reticulum and sarcoplasmicreticulum (SR; a specialized version of the endoplasmic reticulum instriated muscle) storage organelles is achieved throughsarcoplasmic-endoplasmic reticulum calcium ATPases (SERCAs), commonlyreferred to as calcium pumps. During signaling (i.e., when endoplasmicreticulum channels are activated to provide for calcium release from theendoplasmic reticulum into the cytoplasm), endoplasmic reticulum calciumis replenished by the SERCA pump with cytoplasmic calcium that hasentered the cell from the extracellular medium (Yu and Hinkle (2000) J.Biol. Chem. 275:23648-23653; Hofer et al. (1998) EMBO J. 17:1986-1995).

Calcium release channels associated with IP₃ and ryanodine receptorsprovide for controlled release of calcium from endoplasmic andsarcoplasmic reticulum into the cytoplasm resulting in transientincreases in cytoplasmic calcium concentration. IP₃ receptor-mediatedcalcium release is triggered by IP₃ formed in the break down of plasmamembrane phosphoinositides through the action of phospholipase Cactivated by binding of an agonist to a plasma membrane Gprotein-coupled receptor. Ryanodine receptor-mediated calcium release istriggered by an increase in cytoplasmic calcium and is referred to ascalcium-induced calcium release (CICR). The activity of ryanodinereceptors (which have affinity for ryanodine and caffeine) may also beregulated by cyclic ADP-ribose.

Thus, the calcium levels in the stores, and in the cytoplasm, fluctuate.For example, ER free calcium can decrease from a range of about 60-400μM to about 1-50 μM when HeLa cells are treated with histamine, anagonist of PLC-linked histamine receptors (Miyawaki et al. (1997) Nature388:882-887). Store-operated calcium entry is activated as the freecalcium concentration of the intracellular stores is reduced. Depletionof store calcium, as well as a concomitant increase in cytosolic calciumconcentration, can thus regulate store-operated calcium entry intocells.

ii. Store-Operated Ionic Currents

Electrophysiological analysis of store-operated or calciumrelease-activated currents reveals distinct biophysical properties (see,e.g., Parekh and Penner (1997) Physiol. Rev. 77:901-930) of thesecurrents. For example, the current can be activated by depletion ofintracellular calcium stores (e.g., by nonphysiological activators suchas thapsigargin, CPA, ionomycin and BAPTA, and physiological activatorssuch as IP₃) and can be selective for divalent cations, such as calcium,over monovalent ions in physiological solutions or conditions, can beinfluenced by changes in cytosolic calcium levels, and can show alteredselectivity and conductivity in the presence of low extracellularconcentrations of divalent cations. The current may also be blocked orenhanced by 2-APB (depending on concentration) and blocked by SKF96365and Gd3+ and generally can be described as a calcium current that is notstrictly voltage-gated.

c. Cytoplasmic Calcium Buffering

Agonist activation of signaling processes in cells can involve dramaticincreases in the calcium permeability of the endoplasmic reticulum, forexample, through opening of IP₃ receptor channels, and the plasmamembrane through store-operated calcium entry. These increases incalcium permeability are associated with an increase in cytosoliccalcium concentration that can be separated into two components: a“spike” of calcium release from the endoplasmic reticulum duringactivation of the IP₃ receptor and a plateau phase which is a sustainedelevation of calcium levels resulting from entry of calcium into thecytoplasm from the extracellular medium. Upon stimulation, the restingintracellular free calcium concentration of about 100 nM can riseglobally to greater than 1 μM. The cell modulates these calcium signalswith endogenous calcium buffers, including physiological buffering byorganelles such as mitochondria, endoplasmic reticulum and Golgi.Mitochondrial uptake of calcium through a uniporter in the innermembrane is driven by the large negative mitochondrial membranepotential, and the accumulated calcium is released slowly throughsodium-dependent and -independent exchangers, and, under somecircumstances, the permeability transition pore (PTP). Thus,mitochondria can act as calcium buffers by taking up calcium duringperiods of activation and slowly releasing it later. Uptake of calciuminto the endoplasmic reticulum is regulated by the sarcoplasmic andendoplasmic reticulum calcium ATPase (SERCA). Uptake of calcium into theGolgi is mediated by a P-type calcium transport ATPase (PMR1/ATP2C1).Additionally, there is evidence that a significant amount of the calciumreleased upon IP₃ receptor activation is extruded from the cell throughthe action of the plasma membrane calcium ATPase. For example, plasmamembrane calcium ATPases provide the dominant mechanism for calciumclearance in human T cells and Jurkat cells, although sodium/calciumexchange also contributes to calcium clearance in human T cells. Withincalcium-storing organelles, calcium ions can be bound to specializedcalcium-buffering proteins, such as, for example, calsequestrins,calreticulins and calnexins. Additionally, there are calcium-bufferingproteins in the cytosol that modulate calcium spikes and assist inredistribution of calcium ions. Thus, proteins and other molecules thatparticipate in any of these and other mechanisms through which cytosoliccalcium levels can be reduced are proteins that are involved in,participate in and/or provide for cytoplasmic calcium buffering.

d. Receptor-Mediated and Second Messenger-Operated Cation Movement

Receptor-mediated cation channels are gated in response to ligandbinding to a membrane receptor distinct from the channel protein itself.Some receptor-mediated cation channels are activated downstream oftyrosine kinases and others via G protein signaling cascades.Receptor-mediated channels are expressed in a number of both excitableand nonexcitable cells, including smooth muscle, mast cells, epidermisand renal mesangial cells.

One way in which receptor-mediated cation channels are regulated isthrough second messengers induced in response to ligand-binding to amembrane receptor. Such cation channels are referred to as secondmessenger-operated channels. For example, cyclic nucleotides generatedby adenylyl and guanylyl cyclases can directly activate cation-permeablechannels. Such cyclic nucleotide-gated channels are predominantlyexpressed in sensory tissues, for example the retina and inolfactory/gustatory epithelia. Calcium is another second messenger thatcan mediate ion channel function. Examples of calcium-mediated channelsinclude calcium-activated potassium and chloride channels as well ascation channels in neutrophils, smooth muscle and mast cells.

Inositol phosphates generated upon activation of phospholipase C (PLC)can also act as second messengers that activate certain channels. Forexample, channels responsive to inositol-1,4,5-triphosphate (IP₃)include the intracellular IP₃ receptor of the endoplasmic reticulum aswell as plasma membrane channels such as those expressed inT-lymphocytes, mast cells and epidermal cells. The intracellular IP₃receptor functions as a ligand-gated ion channel that permits passage ofcalcium upon binding of IP₃ released through hydrolysis of membranephospholipids by activated phospholipase C (PLC). PLC can be activatedthrough agonist binding to a surface membrane G protein-coupledreceptor. Activation of the IP₃ receptor results in the release ofcalcium stored in the endoplasmic reticulum into the cytoplasm whichproduces a transient “peak” increase in cytosolic calcium concentration.In addition, although no cation channels have been identified incomponents of the endocytic pathway, e.g., endosomes and lysosomes,IP₃-dependent agonists appear to be associated with calcium release fromlysosomes in MDCK cells (see Haller et al. (1996) Biochem. J.319:909-912).

Lipids and polyunsaturated fatty acids (PUFAs) may also act as secondmessengers for the activation of ion channels. For example, arachidonicacid and its metabolites, as well as linolenic acid, can activatereceptor-mediated ion channels. In addition, phospholipids, such aslysophospholipids (e.g., lysophosphatidic acid (LPA),lysophosphatidylcholine (LPC), sphingosylphosphoryl choline (SPC) andsphingosine 1-phosphate (SIP)) can be ligands for plasma membranereceptors such as G-protein-coupled receptors (GPCRs) involved in asecond messenger cascade process in cells (see, e.g., Hla et al. (2001)Science 294:1875). PLC generates not only IP₃ but also diacylglycerol(DAG) which is a potential precursor for polyunsaturated fatty acids.PUFAs can be released from DAG by the action of DAG lipase.

e. Calcium Uptake and Release by Endosomes and Lysosomes

Endocytosis is a process whereby contents of the cell plasma membraneand extracellular medium are transported into the interior of the cell.Not only does endocytosis serve “house-keeping” functions of a cell, itplays crucial roles in cell signaling, development, and the regulationof varied biological processes including, for example, synaptogenesis,neural plasticity, generation of morphogen gradients and programmed celldeath. Endocytosis may have both negative and positive influences onsignaling. For example, endocytosis can regulate the number of receptorson the plasma membrane. In addition, endocytosis plays a positive rolein signaling mediated by the Notch signaling pathway which acts todetermine cell-type specificity during development.

The endocytic process involves several components, including endosomesand lysosomes which are intracellular compartments along the endocyticpathway. Endosomes are morphologically heterogeneous and constitute apleiomorphic smooth membrane system of tubular and vesicular elements.The vesicular elements contain intra-organelle vesicles and aredescribed as multivesicular bodies. Endocytosed macromolecules aredelivered first to early endosomes and then to late endosomes. Earlyendosomes are tubular with varicosities and many are locatedperipherally within the cell close to the plasma membrane. Lateendosomes are more spherical and have the appearance of multivesicularbodies. They are mostly juxtanuclear being concentrated near themicrotubule organizing center. Early and late endosomes arecharacterized by different lumenal pHs, different protein compositionsand association with different small GTPases of the Rab family. Theearly endosome is the major sorting compartment of the endocytic pathwaywhere many ligands dissociate from their receptors and from which manyof the receptors recycle to the cell surface.

Lysosomes are membrane-bound organelles containing hydrolytic enzymesand are regarded as the terminal degradation compartment of theendocytic pathway. Lysosomes also play an important role inphagocytosis, autophagy, crinophagy and proteolysis of some cytosolicproteins that are transported across the lysosomal membrane. In manycell types, lysosomes secrete their contents after fusion with theplasma membrane. The limiting membrane of lysosomes contains a set ofhighly glycosylated lysosomal-associated membrane proteins (LAMPs).Additional lysosomal membrane proteins mediate transport of ions, aminoacids, and other solutes across the lysosomal membrane.

In mammalian cells, the organelles of the late endocytic pathwayinteract with each other and are in dynamic equilibrium. Content mixingand/or exchange of membrane proteins occurs between late endosomes,lysosomes and between late endosomes and lysosomes. Delivery ofendocytosed macromolecules to lysosomes occurs by content mixing betweenlate endosomes and lysosomes as a result of transient as well as directfusion which can form hybrid organelles. Cell-free heterotypic fusion ofmammalian late endosomes and lysosomes requires calcium which maymediate its effects via calmodulin. The calcium is derived and releasedfrom the endocytic organelle lumen and is required in a late step infusion. Although the calcium release pathway has not yet beenelucidated, docking of endocytic organelles is thought to triggerrelease of endocytosed calcium from the lumen of endocytic organellesinto the cytoplasm. This calcium release is believed to mediate membranefusion events at several stages on the endocytic pathway. Lysosomescontain a mobilizable calcium pool. Furthermore, although no cationtransport molecule has been identified in endosomes or lysosomes, bothendosomal and lysosomal membranes provide for calcium transport. Thus,uptake and release of calcium from endosomes and lysosomes can impactintracellular and cytosolic calcium levels.

2. Molecules Involved in Intracellular Calcium Modulation andCalcium-Entry Mediated Events

Fluctuations in the level of calcium ions, including free and boundcalcium, in cells provide important biological signals involved inprocesses such as, for example, protein secretion, muscle contraction,cell death and development. The movement of cations, such as calcium,into, within and out of cells thus plays a critical role in theoperation and survival of cells. Calcium binding and movement into, outof and within cells can act as a signal that is highly organized inspace, frequency and amplitude due in part to localization of themovements and tight regulation of the processes through which calciummovement occurs in cells.

Altered calcium regulation or calcium dyshomeostasis or dysregulation incells is associated with a number of diseases and disorders. Alteredcalcium regulation can be the result of alterations in the elementsinvolved in movement of calcium ions, and/or in the regulation thereof.It is, therefore, desirable to identify cellular constituents, such asproteins, that modulate intracellular calcium, including, for example,proteins that provide for and/or regulate the movement of cations, suchas calcium, into, within and out of cells, and to identify agents thatmodulate intracellular calcium. Identification of molecules, e.g.,proteins, involved in modulating intracellular calcium makes possiblethe elucidation of the molecular and cellular mechanisms underlyingmodulatory processes such as calcium binding and movement. An example ofone such process is store-operated calcium entry, a fundamental andessential process of many cells. The identities of the molecularcomponents that are involved in, participate in and/or provide forstore-operated calcium entry, and, in particular, the identities of theion transport proteins or channels that provide for store-operatedcalcium entry, are largely unknown and may differ for many cell types.Identification of such molecules provides molecular targets for theregulation of this specific and critical process in calcium-dependentcellular functions.

Furthermore, identification of molecules involved in modulatingintracellular calcium assists in the dissection of complex signalingprocesses and facilitates the elucidation of the elements involved inregulation of these processes. Such processes include, but are notlimited to, receptor-mediated, store-operated, and secondmessenger-operated cation entry into the cytoplasm or intracellularorganelles. Knowledge of the number and structure of such molecules, aswell as a comparison of their properties, permits the identification anddesign of agents that specifically interact with and/or affect orregulate molecules, such as, for example, ion transport proteins, thatmodulate intracellular calcium. Such agents have many uses. For example,they can be used to assess function and distribution of proteins thatmodulate intracellular calcium. The specific identified proteins mayalso be used as targets in methods for identifying agents that modulateintracellular, such as cytosolic, calcium and candidate therapeuticagents. Furthermore, the identified proteins, as well as nucleic acidsencoding the proteins, may be used to modulate intracellular calcium,for example, by recombinant expression of nucleic acid encoding such aprotein in a cell, or by reducing, altering, eliminating or interferingwith expression of such a protein in a cell.

Molecules that can be involved in modulating intracellular calciuminclude, but are not limited to, calcium-binding proteins, ion transportproteins that are involved in providing for movement of cations, such ascalcium, into, out of or within cells, receptors and proteins thatregulate ion transport proteins, receptors or calcium-binding proteins.A molecule involved in modulating intracellular calcium can do so at anylevel and in connection with any of a number of processes within a cell.For example, a molecule involved in modulating intracellular calcium mayparticipate in the maintenance of resting cytosolic calcium levels,store-operated calcium entry into cells, receptor-mediated calciummovement, second messenger-operated calcium movement, calcium influxinto or efflux from a cell, and/or calcium uptake into or release fromintracellular compartments, including, for example, the endoplasmicreticulum, endosomes and lysosomes. A molecule involved in modulatingintracellular calcium may function alone (e.g., as a single unit or as ahomo-multimer of two or more of proteins) or in combination with othermolecules, e.g., proteins (e.g., in a heteromeric configuration), andmay be involved in regulating proteins that bind and/or transportcalcium or receptors, particularly receptors involved inreceptor-mediated cation movement or cell signaling.

a. Ion Transport Proteins

Ion transport proteins are proteins involved in providing for thetransport of ions into, within, or out of cells. Ion transport proteinsinvolved in modulating intracellular calcium are involved in providingfor the transport of calcium. Such ion transport proteins may berelatively specific for calcium ion transport.

i. Structural Features of Ion Transport Proteins

An ion transport protein may function to transport cations in a numberof ways. For example, the proteins may form and/or contribute to acomplex that forms a pore or channel for the transport of cationsthrough a membrane. The proteins may instead provide for a translocationof cations through ion binding and release processes as ischaracteristic of a transporter. Ion transport proteins may function totransport ions as a single unit or may be one unit of a multi-componentstructure that transports ions. A multi-component structure may be ahomo-multimer of two or more of the same proteins or a hetero-multimercontaining two or more different proteins (and which may also containtwo or more of the same proteins).

Ion transport proteins involved in providing for movement of ions via achannel-like or pore structure typically contain one or multiple regionsthat have characteristics of transmembrane domains, which may possiblyparticipate in channel formation. Transmembrane domains tend to includea sequence of amino acids, typically of about 10 to about 30 or moreamino acids, about 15 to about 30 or more amino acids, about 20 to about30 or more amino acids, or about 20 to about 25 amino acids, of highhydrophobicity. The hydrophobicity scale is defined from the transferfree energy of amino acids between organic solvents and water, andstatistics on the distribution of residues in proteins. Hydropathy plotsof the hydrophobicity of adjacent amino acid residues averaged over amoving window of suitable length are commonly used to assess proteinsfor such sequences of amino acids (see, e.g., Kyte and Doolittle (1982)J. Mol. Biol. 157:105-134; Argos et al. (1982) Eur. J. Biochem.128:565-575 and Engelman et al. (1984) Ann. Rev. Biophys. Biophys. Chem.15:321-353). Although transmembrane domains may be in an α-helicaland/or β-sheet conformation, the transmembrane domain structure of mostmajor families of ion channel proteins appears to be α-helical.

Pore-lining segments of ion transport protein channel transmembranedomains, such as regions lining aqueous channels, may have a partlyhydrophilic face and appear as amphipathic segments. Amphipathicsegments may be identified based on the hydrophobic moment (see, e.g.,Eisenberg et al. (1984) J. Mol. Biol. 179:125-142; and Finer-Moore andStroud (1984) Proc. Natl. Acad. U.S.A. 81:155-159).

The number of transmembrane domains contained in an ion transportprotein that includes a channel-like structure can vary. For example,there can be at least one transmembrane domain contained in a proteininvolved in providing for ion transport. Thus, for example, there can beone, two, three, four, five, six or more transmembrane domains containedin a protein involved in providing for ion transport. Typically, thereare at least about 1 to about 25 or more transmembrane domains, about 2to about 25 or more transmembrane domains, about 4 to about 25 or moretransmembrane domains, about 6 to about 25 or more transmembranedomains, about 8 to about 25 or more transmembrane domains, or about 1or 2 to about 8 transmembrane domains or about 6 or more transmembranedomains. For example, an ion transport protein that includes achannel-like structure and is capable of providing for the movement ofcations through a membrane may contain at least one, or at least two, orat least three or at least 4 or more groups of six transmembranehelices.

Multimeric complexes form the complete structure of many ion transportchannels. These complexes can contain two or more different subunits.Typically, at least one subunit of a complex is designated the poresubunit, which may be able to flux ions in the absence of othersubunits. Other subunits of such complexes, which alone may not be ableto flux ions, are typically required for normal kinetics and modulation,e.g., gating, activation, and deactivation) of the channel. Suchsubunits have been referred to as auxiliary subunits of a channelcomplex. For example, voltage-gated sodium channels contain an αsubunit, which has a functional pore sufficient for functionalexpression of sodium currents, and two auxiliary subunits, β1 and β2subunits, which are required for normal kinetics and thevoltage-dependence of gating of sodium channels. The α subunit is largerand contains multiple domains containing multiple α-helicaltransmembrane segments, whereas the β subunits have a singletransmembrane segment, a large, glycosylated extracellular domain and asmall intracellular domain (see, e.g., Catterall (2000) Neuron26:13-25).

ii. Calcium Transport

Ion transport proteins that modulate intracellular calcium are involvedin the transport of calcium. Calcium transport may be assessed in avariety ways. For example, cells expressing an ion transport protein maybe evaluated for uptake of labeled calcium, such as 45Ca²⁺, into thecells. RNA coding for an ion transport protein may also be introducedinto a cell, e.g., Xenopus laevis oocytes, which may be evaluated for⁴⁵Ca²⁺ uptake.

Calcium transport properties of an ion transport protein may also beassessed using calcium indicator-based assays of the intracellularcalcium levels of cells expressing the protein. Such assays utilizecalcium-sensitive indicators which facilitate detection of transientalterations in intracellular calcium levels. These indicators provide adetectable signal, e.g., fluorescence or bioluminescence, upon bindingof calcium and therefore can be correlated to calcium levels in cells.Methods of measuring intracellular calcium using calcium indicators arewell known in the art (see, e.g., Takahashi et al. (1999) Physiol. Rev.79:1089-1125).

Electrophysiological analysis of cells expressing an ion transportprotein also can be used to assess calcium transport by a transportprotein. For example, whole-cell, patch-clamp, voltage clamp andsingle-channel recording methods may be used to detect and measurecalcium or other cation currents across membranes of cells to whichcalcium or another cation has been applied. A variety of cells may beused for such electrophysiological analysis, including, but not limitedto, X. laevis oocytes into which RNA encoding an ion transport proteinhas been injected.

Ion transport protein(s) involved in calcium regulation in cells may bemore permeable to calcium than to monovalent ions under physiologicalconditions. Thus, for example, such types of proteins can be able toflux calcium through a membrane more readily than monovalent ions. Forexample, the permeability of an ion transport protein to calcium may bemore than about 1.5-fold, more than about 2-fold, more than about3-fold, more than about 4-fold, more than about 5-fold, more than about6-fold, more than about 7-fold, more than about 8-fold, more than about9-fold or more than about 10-fold greater than the permeability tomonovalent ions. Ion transport proteins may be selective for calciumunder physiological conditions.

b. Modulatory or Regulatory Molecules

In addition to ion transport proteins, there may be cellular moleculesthat are involved in modulating intracellular calcium but that are notdirectly involved in providing for the transport of ions into or out ofthe cytosol as would, for example, a channel protein or pore-formingsubunit of a channel protein. Such molecules, which can be referred toas modulatory or regulatory molecules (i.e., modulators or regulators),encompass a diverse array of structures and include receptors, enzymesand calcium-binding proteins. Modulator or regulator molecules can, forexample, function in processing a signal in a cell (e.g., signalingpathway components) or regulate the activity of an enzyme or ionchannel.

For example, some calcium-binding proteins can be involved in modulatingintracellular calcium. Included among the calcium-binding proteins areproteins that contain a calcium binding motif referred to as an EF-hand(see, e.g., Kretsinger (1997) Nat. Struct. Biol. 4:514-516; Ikura (1996)Trends Biochem. Sci. 21:14-17; Kawasaki et al. (1995) Protein Profile2:297-490). Typically, an EF-hand motif contains a loop of about 12amino acid residues flanked on either side by an alpha helix of about 12amino acid residues. Position 12 of the loop typically contains a Glu orAsp. The helix-loop-helix motif can be repeated from about 2 to about 12times. The motifs may coordinate calcium to side-chain oxygens ofinvariant residues occupying positions 1, 3, 5 and 12 of the loop, andto a carbonyl oxygen of a less conserved residue at position 7. EFhand-containing proteins may undergo a conformational change uponbinding calcium, and thus some of these proteins can pass signalinginformation on to targets to which such proteins can bind. EF handproteins can function in a number of ways. For example, they canfunction as a separate subunit of a single protein (e.g., enzyme), as asubunit that reversibly associates with different proteins (e.g.,calmodulin) or as an integral portion of the sequence of an enzyme(e.g., calpain).

Neuronal calcium sensor (NCS) proteins are examples of proteinscontaining EF hand motifs. These proteins are expressed predominantly orsolely in retinal photoreceptors or neurons. Five subfamilies of NCSproteins have been described: two expressed in retinal photoreceptors(recovering, which inhibit rhodopsin kinase, and the guanylatecyclase-activating proteins (GCAPs)) and three expressed in centralneurons and neuroendocrine cells (frequenins, visinin-like proteins andthe Kv channel-interacting proteins) (see, e.g., Burgoyne and Weiss(2002) Biochem. J. 353:1-12). The latter three subfamilies may regulateneurotransmitter release, polyphosphoinositide biosynthesis, cyclicnucleotide metabolism and the activity of type A potassium channels. TheNCS proteins undergo conformational changes upon binding calcium andhave thus been referred to as calcium sensors or switches. Most NCSproteins are N-terminally myristoylated, and, after binding of calcium,the myristoyl residue and hydrophobic portions of the sequence areexposed favoring interaction with membranes or target proteins.

Another family of EF hand-containing proteins is the calpain family ofintracellular cysteine proteases. These proteins modulate biologicalactivities of their substrates by limited proteolysis. The protease coreregion of the μ isoform of calpain appears to also contain two non-EFhand calcium-binding sites. Binding of calcium at these sites aligns theactive site cleft and converts the core into an active enzyme withcalpain-like specificity (see, e.g., Moldoveanu et al. (2002) Cell108:649-660). Thus, there are at least three different types ofcalcium-binding sites in calpains: EF-hand, C2-like domain and proteasedomain sites.

Another example of an EF hand protein is calmodulin (CaM). Calmodulininteracts with numerous diverse proteins, including, for example,CaM-dependent serine/threonine kinases, CaM kinase II, CaM kinase kinaseand myosin light chain kinase, calcineurin, CaM-calcium-activatedpotassium channel and anthrax adenylyl cyclase. The roles of CaM bindingto such target proteins include, for example, release of autoinhibitorydomains of kinases, CaM tethering and calcium-dependent inactivation ofthe target protein, active site remodeling of adenylyl cyclase,dimerization and activation of channel proteins. CaM recruitment motifsof CaM-binding domains of CaM-dependent proteins have been predicted(see, e.g. Rhoads and Friedberg (1997) FASEB J. 11:331-340; Hoeflich andIkura (2002) Cell 108:739-742 and calcium.uhnres.utoronto.ca). Anexample of a CaM-binding motif is an “IQ motif” having the followingconsensus sequence which can be present in tandem repeats: IQXXXRGXXXR.

c. Downstream Calcium Entry-Mediated Events

In addition to intracellular changes in calcium stores, store-operatedcalcium entry affects a multitude of events that are consequent to or inaddition to the store-operated changes. For example Ca²⁺ influx resultsin the activation of a large number of calmodulin-dependent enzymesincluding the serine phosphatase calcineurin. Activation of calcineurinby an increase in intracellular calcium results in acute secretoryprocesses such as mast cell degranulation. Activated mast cells releasepreformed granules containing histamine, heparin, TNFα and enzymes suchas β-hexosaminidase. Some cellular events, such as B and T cellproliferation, require sustained calcineurin signaling, which requires asustained increase in intracellular calcium. A number of transcriptionfactors are regulated by calcineurin, including NFAT (nuclear factor ofactivated T cells), MEF2 and NFκB. NFAT transcription factors playimportant roles in many cell types, including immune cells. There arefour calcium-regulated members of the NFAT family. In immune cells NFATmediates transcription of a large number of molecules, includingcytokines, chemokines and cell surface receptors. Transcriptionalelements for NFAT have been found within the promoters of cytokines suchas IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, as well as tumor necrosis factoralpha (TNFa), granulocyte colony-stimulating factor (G-CSF), andgamma-interferon (γ-IFN).

The activity of NFAT proteins is regulated by their phosphorylationlevel, which in turn is regulated by both calcineurin and NFAT kinases.Activation of calcineurin by an increase in intracellular calcium levelsresults in dephosphorylation of NFAT and entry into the nucleus.Rephosphorylation of NFAT masks the nuclear localization sequence ofNFAT and prevents its entry into the nucleus. Because of its strongdependence on calcineurin-mediated dephosphorylation for localizationand activity, NFAT is a sensitive indicator of intracellular calciumlevels.

C. Identification of Molecules Involved in Modulating IntracellularCalcium

Described herein is the identification of cellular proteins involved inmodulating intracellular calcium. Proteins identified herein as beinginvolved in modulating intracellular calcium may participate in one ormore of a number of processes that affect intracellular calcium,including, but not limited to, receptor-mediated or secondmessenger-operated calcium movement, calcium uptake and/or release byintracellular organelles (e.g., endoplasmic reticulum, endosomes andlysosomes), cytosolic calcium buffering and store-operated calcium entryinto cells. A direct participation of a protein in a particular processor pathway not only affects that process but can also indirectly affectone or more other processes or pathways. Thus, for example, a proteinthat has a direct role in a G-protein-coupled receptor signaling pathwaymay also indirectly affect store-operated calcium entry into a cell byaffecting the release of calcium from the endoplasmic reticulum, whichis involved in providing a signal for calcium entry via store-operatedion channels.

Proteins identified as being involved in modulating intracellularcalcium include, but are not limited to, proteins that are homologous toa protein encoded by a Drosophila or mammalian (e.g., human or rodentsuch as rat, hamster or mouse) gene that, when altered in its expressionin a cell, results in altered intracellular calcium. The protein can beone that is homologous to a protein encoded by a Drosophila or mammaliangene that, when altered in its expression, results in alteredstore-operated calcium entry, altered calcium levels in, or movement ofcalcium into, out of or within an intracellular organelle or calciumstore (e.g., endoplasmic reticulum), altered cytoplasmic calciumbuffering and/or altered basal cytosolic calcium levels. The identifiedcellular proteins include mammalian proteins, e.g., rodent and humanproteins, nematode, e.g., C. elegans, proteins and insect, e.g.,Drosophila, proteins. The proteins include modulatory proteins,receptors and proteins that are involved in, participate in and/orprovide for the movement of calcium, such as, for example, an iontransport protein, or a component of an ion transport protein complex,that is involved in, participates in and/or provides for store-operatedcalcium entry.

Also provided herein are methods for identifying additional elements(e.g., proteins and other cellular or cell-associated molecules)involved in modulating intracellular calcium, identifying agents thatmodulate intracellular calcium and methods for modulating intracellularcalcium and for treating diseases and disorders. Such methods canutilize or target the proteins identified herein as being involved inmodulating intracellular calcium, such as, for example, by modulating orby participating in store-operated calcium entry.

1. Stromal Interacting Molecule (STIM) Proteins

As described herein, proteins identified as being involved in modulatingintracellular calcium include stromal interacting molecules (alsoreferred to as STIM, SIM and GOK) or STIM-like proteins or portionsthereof. Proteins identified herein as proteins involved in modulatingintracellular calcium include proteins homologous to a protein encodedby the coding sequence of Drosophila gene CG9126 (GenBank Accession no.NM_(—)078633, gi22832319 and AF328906; see also SEQ ID NO. 1 for genecoding sequence and SEQ ID NO. 2 and GenBank Accession no. NP_(—)523357,AAF48542, AAK82338 and P83094 for amino acid sequence) and/or amammalian stromal interacting molecule (STIM) protein (see, e.g., SEQ IDNO: 90 for a mammalian STIM1 consensus amino acid sequence), such as,for example, human STIM1 (GenBank protein Accession nos. Q13586,NP-003147, AAC51627 and nucleotide Accession nos. NM_(—)003156,gi2264345, gi2264346; see also SEQ ID NOS:82 and 3 for nucleic acidcoding sequences and SEQ ID NOS:84, 83 and 4 for amino acid sequences),Chinese hamster STIM1 (see SEQ ID NO: 95 for a partial nucleic acidcoding sequence and SEQ ID NO: 96 for a partial amino acid sequence),rat STIM1 (see SEQ ID NO: 97 for a partial nucleic acid coding sequenceand SEQ ID NO: 98 for a partial amino acid sequence) and also referenceSTIM sequence. (see SEQ ID NO: 51 for a nucleic acid coding sequence andSEQ ID NO: 52 for an amino acid sequence). As described herein, suchproteins have been identified as being involved in, participating inand/or providing for store-operated calcium entry or modulation thereof,cytoplasmic calcium buffering and/or modulation of calcium levels in ormovement of calcium into, within or out of intracellular calcium stores(e.g., endoplasmic reticulum). Particular proteins include stromalinteracting molecule (STIM) proteins and STIM-like proteins, including,but not limited to, mammalian STIM-1, such as human and rodent (e.g.,mouse) STIM-1, Drosophila melanogaster D-STIM, C. elegans C-STIM,Anopheles gambiae STIM and mammalian STIM-2, such as human and rodent(e.g., mouse) STIM-2. Proteins identified herein as being involved inmodulating intracellular calcium include proteins that are at leastabout 45% homologous to the protein encoded by the coding sequence ofDrosophila gene CG9126 and/or a mammalian stromal interacting molecule(STIM) protein over at least about 52% of the protein.

STIM proteins are type I transmembrane phosphoproteins with a singletransmembrane segment separating the extracellular portion from thecytosolic domain. Vertebrates have two forms of the STIM protein, STIM1and STIM2, while invertebrates have only one genetic form, e.g., D-STIMin Drosophila melanogaster. Comparison of the vertebrate andinvertebrate STIM proteins demonstrates a conserved genomicorganization, indicating that the two STIM genes found in vertebrateslikely arose from a single ancestral gene (Williams et al. (2001)Biochem. J. 357: 673-685).

STIM proteins include, but are not limited to, STIM1 (SEQ ID NO. 4 forHomo sapiens, SEQ ID NO: 52 for reference STIM, and SEQ ID NO. 16 forMus musculus) and STIM2 (SEQ ID NO. 6 for Homo sapiens and SEQ ID NO. 28for Mus musculus) in humans and other vertebrates, D-STIM (SEQ ID NO. 2)in Drosophila melanogaster, and C-STIM in Caenorhabditis elegans (SEQ IDNOs. 24 and 26). Partial sequences for STIM proteins include SEQ ID NO.98 (rat) and SEQ ID NO. 96 (hamster). Other potential orthologs of STIMproteins were identified in Tblastn searches of GenBank databases.Expressed sequence tags (ESTs) were identified for STIM1 genes in rat(Rattus norvegicus; Genbank accession nos. AA996745 and AI763957),bovine (Bos taurus; Genbank accession nos. AV609285 and AW669469) andswine (Sus scrofa; Genbank accession nos. AW787215 and BE663170). ESTsrepresenting STIM2 homologs were identified in mouse (Mus musculus;Genbank accession nos. AA088943, AI194208, AW106055, AW910374, BE652414and BF463756), rat (Rattus norvegicus; Genbank accession nos. AA944338,BF286659 and BF296660), bovine (Bos taurus; Genbank accession no.BE482998), amphibian (Xenopus laevis; Genbank accession nos. AW633493,AW639117 and BF427891) and avian (Gallus gallus; Genbank accession no.AI981296).

Sources of nucleic acid molecules encoding STIM proteins in humansinclude human tissues, cancerous cells and cell lines, including, butnot limited to, prostate; uterus; colon; testis; kidney, breast;placenta; heart; mammary gland; muscle (skeletal); lung; whole brain;marrow; ovary; nervous; liver; fetal eyes; sciatic nerve; cerebellum;pooled adenocarcinoma with signet ring cell features; pooled humanmelanocyte, fetal heart, and pregnant uterus; B-cell, chronic lymphoticleukemia; lymphoma, follicular mixed small and large cell; thymus,pooled; anaplastic oligodendroglioma; adenocarcinoma; serous papillarycarcinoma; carcinoma in situ from retromolar trigone; renal cell tumor;glioblastoma; corresponding non cancerous liver tissue; adenocarcinoma(cell line); follicular carcinoma; large cell carcinoma,undifferentiated; melanotic melanoma; senescent fibroblast; epithelioidcarcinoma; cervical carcinoma (cell line); choriocarcinoma; prostatetumor; anaplastic oligodendroglioma with 1p/19q loss; neuroblastoma;renal carcinoma (ascites); lymphoma; nervous tumor; melanocyte; lungtumor; renal cell adenocarcinoma; neuroblastoma (cell line); primaryB-cells from tonsils (cell line); embryonal carcinoma (cell line);melanotic melanoma, high MDR (cell line); epithelioid carcinoma (cellline); Islets of Langerhans; leiomyosarcoma; natural killer cells (cellline); fetal eyes, lens, eye anterior segment, optic nerve, retina,Retina Foveal and Macular, RPE and Choroid; leukopheresis; colon tumor;parathyroid tumor; ascites; astrocytoma grade IV, cell line; PrimaryLung Cystic Fibrosis Epithelial Cells; Lung Focal Fibrosis;Chondrosarcoma Grade II; duodenal adenocarcinoma (cell line); epidermoidcarcinoma (cell line); teratocarcinoma (cell line);moderately-differentiated endometrial adenocarcinoma, 3 pooled tumors;metastatic prostate bone lesion; pooled germ cell tumors; schizophrenicbrain S-11 frontal lobe; kidney tumor; and carcinoid.

The STIM1 gene (also referred to as GOK) has been identified as beingpart of a set of genes that maps to the human chromosome region 11p15.5which is associated with adult and childhood tumors (Manji et al. (2000)Biochem. Biophys. Acta 1481: 147-155; Sabbioni et al. (1999) Cytogenet.Cell Genet. 86: 214-218). Functional studies have indicated that the11p15 region includes tumor and growth suppressive functions (Dowdy etal. (1991) Science 254: 293-295; Koi et al. (1993) Science 260: 361-364;Negrini et al. (1994) Cancer Res. 55: 2904-2909; Reid et al. (1996) Hum.Mol. Genet. 5: 239-247; Satoh et al. (1993) o: 157-164), and geneticstudies have identified at least three regions of loss of heterozygositywithin the chromosome band (Negrini et al. (1995) Cancer Res. 55:3003-3007). Transfection experiments have shown that STIM1 may act as agrowth suppressor in the rhabdomyoscarcoma and rhabdoid tumor cell linesRD and G401 (Sabbioni et al. (1997) Cancer Res. 57: 4493-4497). Theexpression of STIM1 within these cells lines induced growth arrest anddegeneration, indicating that down-modulation of STIM1 expression couldbe the mechanism leading to cell immortalization. The STIM1 genepromoter has been identified within a 1.8-kb SacI fragment at the 5′ endof the gene (Sabbioni et al. (1999) Cytogenet. Cell Genet. 86:214-218;see also GenBank Accession no. AF139917 and SEQ ID NO:93). Transienttransfection of RD cells with a vector containing the STIM1 promotersequence upstream of DNA encoding a luciferase reporter molecule(pGL3-STIM1.B; see Sabbioni et al. (1999) Cytogenet. Cell Genet.86:214-218) which had been methylated in vitro results in a reduction ofluciferase activity compared to that of cells transfected withnon-methylated vector.

On the other hand, STIM1 has also been identified as a stromal cellproduct that binds to the surface of hematopoietic cells, such as pre-Blymphoid cells, and can promote proliferation of these cells (Oritaniand Kincade (1996) J. Cell Biol. 134:771-782). Additionally,overexpression of STIM in PC12 (overexpression of both STIM1 and STIM2)cells has been reported to alter cell phenotype in vivo (includingelongated morphology and increased migratory behavior) and generate aphenotype in transgenic Drosophila (overexpressing DSTIM) withsimilarity to Drosophila Delta and Notch mutants indicating anantagonist effect of STIM on the Notch signaling pathway (see PCTApplication publication no. WO02/30976).

a. STIM and STIM-Like Protein Features

Although DSTIM, STIM1 and STIM2 share some conserved features, there arealso areas in which the proteins diverge. The three STIM proteins differin length from about 570 amino acids for the Drosophila STIM protein(including signal peptide) to about 685 amino acids for STIM1 (includingsignal peptide) and about 746 amino acids (including signal peptide) forSTIM2. Each STIM protein contains a transmembrane domain located withinthe first one-third to one-half of the protein which is bounded oneither side by N-terminal and C-terminal portions of the protein. TheN-terminal portion of STIM may be a putative extracellular domain,whereas the C-terminal portion may be a putative cytoplasmic domain.

The N-terminal putative extracellular domains of DSTIM, STIM1 and STIM2contain an N-terminal signal peptide followed by approximately 271, 190and 203 amino acid residues, respectively. The signal peptide of humanSTIM1 and DSTIM, which is cleaved to yield a mature protein, correspondsto the sequence of the first approximately 22-23 amino acids of theprotein (see, e.g., the first 22 amino acids of a human STIM1 amino acidsequence (SEQ ID NO: 4 and the first 23 amino acids of a DSTIM aminoacid sequence (SEQ ID NO: 2). In contrast, the signal peptide of humanSTIM2 contains about 14 amino acids (see the first 14 amino acids of SEQID NO: 6), which is about 60% of the length of the signal peptides ofhuman STIM1 and DSTIM.

The N-terminal domain of all three STIM proteins contains two closelyspaced cysteine residues that can be involved in intra- or inter-chaindisulfide binding (see, e.g., residues corresponding to positions 126and 133 of DSTIM (SEQ ID NO: 2) 49 and 56 of human STIM1 (SEQ ID NO: 4)and 53 and 60 of human STIM2 (SEQ ID NO: 6)). The N-terminal domain ofD-STIM has an additional 70-80 amino acid region (relative to STIM1 andSTIM2 proteins) between the signal peptide and two closely spacedcysteine residues. Beyond the signal sequence for all three proteins,the extracellular domain also includes an EF hand domain (see, e.g.,residues corresponding to positions 155-166 of DSTIM (SEQ ID NO: 2),76-87 of human STIM1 (SEQ ID NO: 4) and 80-91 of human STIM2 (SEQ ID NO:6)) and a sterile α-motif (SAM) domain (see, e.g., residuescorresponding to positions 213-281 of DSTIM (SEQ ID NO: 2), 129-196 ofhuman STIM1 (SEQ ID NO: 4) and 136-204 of human STIM2 (SEQ ID NO: 6)).SAM domains are generally compact five helical bundles with a conservedhydrophobic core and are found in a wide variety of eukaryoticsignaling, scaffolding and adaptor molecules and transcriptionregulators. These domains have also been implicated in mediatingprotein-protein interaction via the formation of homo- and heterotypicoligomers in signaling molecules and transcriptional regulators (Thanoset al. (1996) J. Biol. Chem. 274: 37301-37306). The location of a SAMdomain in the putative extracellular region of STIM proteins appears tobe a unique feature of this family (Williams et al. (2001) Biochem. J.357: 673-685). An N-linked glycosylation site occurs at or very near theN-terminal limit of the SAM domain in all three STIM proteins (see,e.g., residue corresponding to position 212 in DSTIM (SEQ ID NO: 2), 131in human STIM1 (SEQ ID NO: 4) and 135 in human STIM2 (SEQ ID NO: 6)),while STIM1 possesses a unique second N-linked glycosylation site withinthe SAM domain (see, e.g., residue corresponding to position 171 inhuman STIM1 (SEQ ID NO: 4)). The modification of a SAM domain byN-linked glycosylation also appears to be unique to STIM1 (Williams etal. (2002) Biochim. Biophys. Acta 1596:131-137). Another feature thatoccurs in the N-terminal (putative extracellular) region of STIM1 thatis not present in DSTIM or STIM2 is a possible enzymatic cleavage site(see, e.g., residues corresponding to positions 207-209 of human STIM1(SEQ ID NO: 4)). This site, which is a three-amino-acid sequence ofARG-HIS-ASN in STIM1, is reminiscent of other proteolytic sites.

A single predicted transmembrane domain is present in all STIM proteins(see, e.g., residues corresponding to positions 295-312 of DSTIM (SEQ IDNO: 2), 213 (or 214)-234 of human STIM1 (SEQ ID NO: 4) and 218-235 ofhuman STIM2 (SEQ ID NO: 6)) and contains a single cysteine residue.

The C-terminal putative intracellular or cytoplasmic domain isapproximately 451 amino acid residues in length for STIM1 (see, e.g.,residues corresponding to positions 235-685 of human STIM1 (SEQ ID NO:4)), approximately 511 amino acid residues in length for STIM2 (see,e.g., residues corresponding to positions 236-746 of human STIM2 (SEQ IDNO: 6)) and approximately 258 amino acid residues in length for D-STIM(see, e.g., residues corresponding to positions 313-570 of DSTIM (SEQ IDNO: 2)). For STIM1 and STIM2 proteins, this portion of the polypeptidecontains (1) a potential consensus sequence (YYNI) (see, e.g., residuescorresponding to positions 361-364 of human STIM1 (SEQ ID NO: 4) and365-368 of human STIM2 (SEQ ID NO: 6)) for phosphorylation-dependentbinding of Src homology type 2 (SH2) domains, such as, for example, theSH2 domain of the growth factor receptor bound protein 2 (GRB2), (2) twocoiled-coil domains (see, e.g. residues corresponding approximately topositions 238-343 and 362-390 of human STIM1 (SEQ ID NO: 4) and 242-344and 358-394 of human STIM2 (SEQ ID NO: 6)) which is a principal subunitoligomerization motif (typically involving a heptad repeat pattern ofprimarily apolar residues, and (3) a proline- and serine-rich (STIM1;see, e.g., residues corresponding to positions 600-629 of human STIM1(SEQ ID NO: 4)) or proline- and histidine-rich (STIM2; see, e.g.,residues corresponding to positions 533-559 of human STIM2 (SEQ ID NO:6)) domain that contains several potential proline-directedserine/threonine phosphorylation sites (which are potential targets forby members of the proline-directed protein kinase (PDPK) family, such asthe MAP/ERK kinases and cyclin-dependent kinases (Cdk)) and a potentialconsensus binding sequence for 14-3-3 proteins. Also within this generalregion of STIM1 and STIM2 are several potential SH3 domain bindingmotifs, which have a minimum consensus sequence of PXXP (see, e.g., theregion of residues corresponding to positions 573-629 of human STIM1(SEQ ID NO: 4) and 521-559 of human STIM2 (SEQ ID NO: 6)). The two STIMproteins diverge significantly distal to their proline-rich regions withthe exception of lysine-rich tails at their respective C-termini. Thecytoplasmic region of D-STIM is half the length of those of STIM1 andSTIM2. The domain structure of D-STIM is similar to that seen for STIM1and STIM2 in that there is a significant degree of α-helical structure(coiled-coils) (see, e.g., residues corresponding approximately topositions 310-407 and 420-462 of DSTIM (SEQ ID NO: 2)), however, thereappears to be no homologous GRB2 consensus sequence nor a proline-richregion.

The C-terminal putative cytoplasmic region of STIM1 also containsseveral additional domains that are not found in STIM2 and DSTIMproteins. For example, STIM1 contains ATP synthase B/B′ (pfam00430),ezxrin/radixin/moesin (ERM; pfam00769) and diacylglycerol kinaseaccessory (DAGKa; smart00045) domains. The ATP synthase B/B′ domain(see, e.g., residues corresponding approximately to positions 249-337 ofhuman STIM1 (SEQ ID NO: 4)) is a domain that is also found as a part ofthe CF(0) (base unit) of ATP synthase which may translocate protonsthrough membranes (e.g., the mitochondrial inner membrane). STIMproteins contain a domain (referred to as an SMC domain; COG1196) at asimilar region of the C-terminal portion of the protein (see, e.g.,residues corresponding to positions 238-340 of human STIM2 (SEQ ID NO:2)) which is found in Smc chromosome segregation ATPases. Theezrin/radixin/moesin domain (see, e.g., residues correspondingapproximately to positions 253-424 of human STIM1 (SEQ ID NO: 4)) is adomain also found in the ERM family of proteins which contain a band 4.1domain (pfam00373) at the amino terminus. The DAGKa domain (see, e.g.,residues corresponding approximately to positions 422-484 of human STIM1(SEQ ID NO: 4)) which is also found in kinases that are activated bydiacylglycerol (DAG), e.g., protein kinase C.

DSTIM, which has the shortest C-terminal putative cytoplasmic region ofthe STIM proteins, contains a myosin tail domain (see, e.g., residuescorresponding approximately to positions 324-491 of DSTIM (SEQ ID NO:2)) that is not present in STIM1 and STIM2 proteins.

STIM proteins undergo post-translational modification. STIM1 and STIM2are modified by N-linked glycosylation and phosphorylation which occurspredominantly on serine residues. Differing levels of phosphorylation ofSTIM2 may account for two molecular mass isoforms (approximately 105 and115 kDa) of the protein. In contrast, the molecular mass of STIM1 isapproximately 90 kDa, which decreases to about 84 kDa when N-linkedglycosylation is inhibited by tunicamycin. D-STIM (an approximately 65kDa protein), like STIM1 and STIM2, is modified by N-linkedglycosylation (Williams et al. (2001) Biochem. J. 357: 673-685) asevidenced by mobility shift experiments.

b. STIM and STIM-Like Protein Sequences

In particular embodiments of the methods for screening for oridentifying agents and molecules that modulate intracellular calcium, aprotein (and/or nucleic acid, or portion(s) thereof, encoding a protein)used in the method (or on which the method is based) is a STIM, STIM1,STIM2 or STIM-like protein or portion(s) thereof. Examples of suchproteins include the following: STIM1 from Homo sapiens (SEQ ID NO. 4);STIM1 from Mus musculus (SEQ ID NO. 16); D-STIM from Drosophilamelanogaster (SEQ ID NO. 2); C-STIM from Caenorhabditis elegans (SEQ IDNO. 26); STIM2 from Homo sapiens (SEQ ID NO. 6); STIM2 from Mus musculus(SEQ ID NO. 28); and a homolog from Anopheles gambiae str. PEST (SEQ IDNO. 8). In other embodiments, other proteins that may be used in themethods provided herein include, but are not limited to, proteinsinvolved in intracellular calcium modulation that are substantiallyhomologous to the above-listed proteins. These listed proteins and theirsequences exemplify proteins that are useful for methods, materials andsystems provided herein. Naturally occurring and synthesized alternativeforms of these proteins such as allelic forms, isoforms, muteins, andmutated derivatives will have sequence changes that do not abolish theiroperation in intracellular calcium modulation and are useful inembodiments. Mutations and allelic forms of these proteins are known andare contemplated as embodiments.

In a particular embodiment of the methods of screening for oridentifying an agent or molecule that modulates intracellular calcium, aprotein (or nucleic acid, or portion(s) thereof, encoding a protein)involved in modulating intracellular calcium that is used in the method(or on which the method is based) is a STIM or STIM1 protein orportion(s) thereof. In one embodiment of the methods, the protein is onethat is homologous to a DSTIM protein over at least about 77%, or atleast about 78%, or at least about 79% or at least about 80% or at leastabout 85%, or at least about 90%, or at least about 95% or more of theDSTIM protein. For example, the protein can be one that is at leastabout 50%, or at least about 51%, or at least about 52%, or at leastabout 54%, or at least about 55%, or at least about 58%, or at leastabout 60%, or at least about 65%, or at least about 62%, or at leastabout 70% or more homologous to a DSTIM protein over the specifiedextent of the DSTIM protein. In another embodiment, the protein is onethat is homologous to a STIM1 protein (e.g., a mammalian STIM1 protein(see SEQ ID NO: 90 for a mammalian STIM1 consensus sequence of aminoacids), such as a human or rodent STIM1 protein) over at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 92%, or at least about95%, or at least about 97% or more of the STIM1 protein. For example,the protein can be one that is at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about72%, or at least about 75%, or at least about 77%, or at least about80%, or at least about 82%, or at least about 85%, or at least about90%, or at least about 95% or more homologous to a STIM1 protein overthe specified extent of the STIM1 protein.

In another embodiment of the method for screening for or identifying anagent or molecule that modulates intracellular calcium, a protein (ornucleic acid, or portion(s) thereof, encoding a protein) used in themethod (or on which the method is based) is a STIM or STIM-like protein(or portion(s) thereof) that contains one or more or all of thefollowing structural features or sequences: (1) an amino acid sequencecontaining a SAM domain that contains within it one or more N-linkedglycosylation sites and, optionally, an N-linked glycosylation site inthe amino acid sequence on either side of the SAM domain, (2) an aminoacid sequence containing a dibasic sequence that could serve as aproteolytic cleavage site (see, e.g., residues corresponding topositions 207-209 of human STIM1 (SEQ ID NO: 4)), and, in particularembodiments, the consecutive two- or three-amino-acid sequence ofARG-HIS or ARG-HIS-ASN, (3) an amino acid sequence containing a sequencehomologous to an ATP synthase B/B′ domain (pfam00430), (4) an amino acidsequence containing a sequence homologous to an ezxrin/radixin/moesindomain (ERM; pfam00769), and (5) an amino acid sequence containing asequence homologous to a diacylglycerol kinase accessory (DAGKa;smart00045) domain.

2. Rodent STIM Proteins and Nucleic Acid Encoding Rodent STIM Proteins

a. Nucleic Acid Molecules

Provided herein are isolated nucleic acid molecules encoding sequencesfor rodent STIM, including hamster, rat and reference STIM1 sequences(collectively referred to herein as rodent reference STIM and STIMsequences). Also provided are isolated nucleic acid molecules thatencode domains of rodent reference STIM1, or encode polypeptides withsubstitutions relative to rodent reference STIM1 or its domains thatretain at least one biological activity of STIM1 or the domain. Suchnucleic acid molecules generally retain sufficient homology such thatthe resulting encoded polypeptide or domain retains a biologicalactivity.

Also provided are nucleic acid molecules encoding polypeptides with oneor several amino acid substitutions relative to mature reference STIM1,or encoding polypeptides with one or several amino acid substitutionsrelative to various domains of rodent reference STIM1 described herein,which retain at least one biological activity of STIM1 or of theparticular domain. Incorporating amino acid substitutions into rodentreference STIM1 polypeptides and domains may be advantageous, forexample, in enhancing the stability, expression level, immunogenicity,or a biological activity of the polypeptide. Exemplary substitutionsinclude substitutions at positions that differ between mouse and rodentreference STIM1, or between human and rodent reference STIM1, sincethese positions are likely to be tolerant of conservative ornon-conservative substitutions without substantially affecting a STIM1biological activity. Computer programs known in the art that predictthree-dimensional protein structure can also provide guidance inpredicting which amino acid residues can be modified while retaining thefunction of the polypeptide (see, for example, Eroshkin et al. (1993)Comput. Appl. Biosci. 9:491-497).

Such nucleic acid molecules include, but are not limited to, nucleicacid molecules that encode a rodent reference STIM1 that has at least60%, 70%, 80%, 90% or about 95% sequence identity with the rodentreference STIM1s exemplified herein, or that hybridize along their fulllength or along at least about 70%, 80% or 90% of the full length to theexemplified sequence of nucleotides or domain-encoding portion thereof,particularly under conditions of moderate, generally high, stringency.

SEQ ID NO:51 sets forth an exemplary sequence of nucleotides encodingfull-length reference STIM1 (SEQ ID NO:52). SEQ ID NO: 97 sets forth anexemplary sequence of nucleotides encoding a partial rat STIM1. SEQ IDNO: 95 sets forth an exemplary sequence of nucleotides encoding apartial hamster STIM1. Reference STIM contains a fusion of rat andhamster STIM sequences using the partial hamster STIM1 sequence andconstructing a full-length reference sequence using the 5′ and 3′ends ofrat STIM1. It will be appreciated that due to the degeneracy of thegenetic code, the skilled person can select alternative sequences ofnucleotides that encode SEQ ID NOS:51, 95 and 97. The exemplary sequenceof nucleotides set forth in SEQ BD NO:51 differs from SEQ ID NOS:3 and82, which encode a human STIM1, differs from SEQ ID NO:9, which encodesa mouse STIM1 at nucleotide positions 15, 63, 69, 84, 103, 108, 112,120, 150, 183, 201, 207, 210, 213, 240, 300, 303, 312, 330, 357, 402,441, 474, 570, 621, 660, 697, 738, 783, 795, 861, 873, 874, 895, 951,1051, 1062, 1107, 1200, 1224, 1228, 1278, 1299, 1392, 1395, 1452, 1580,1652, 1654, 1675, 1747, 1749, 1173, 1854, 1855, 1881, 1884, 1888, 1896,2001 and 2025 of SEQ ID NO:51. Exemplary of the nucleic acid moleculesencoding full-length reference STIM is that set forth in SEQ ID NO:51,as well as mature reference STIM1.

Also provided are nucleic acid molecules (and the encoded polypeptides)encoding domains of mature rodent reference STIM1, including theextracellular, transmembrane, cytoplasmic, SAM, coiled-coil, Glu-rich,Pro-Ser-rich, ezrin/radixin/moesin (ERM), diacylglycerol kinaseaccessory domain (DAGKa) and Lys-rich domains. Such nucleic acidmolecules can be used in place of nucleic acid molecules encodingfull-length or mature reference STIM1 in many of the applications forSTIM1 described herein. It will be understood that for suchapplications, nucleic acid molecules encoding polypeptides with severaladditional, or several fewer, amino acids at either or both terminirelative to the STIM1 domain boundaries indicated herein will likelyhave equivalent structures and functions, and are thus contemplatedherein.

The nucleic acid molecules can be used, for example, in screeningapplications described herein or to express rodent reference STIM1polypeptides and domains. They also can be used as probes and primers todetect and/or quantify expression of rodent reference STIM1 mRNA in ratand hamster tissues and cell lines. Other applications will be apparentto those skilled in the art.

Also provided are nucleic acid constructs contain the nucleic acidmolecules encoding STIM1 operatively linked to a promoter of geneexpression. The promoter can be a heterologous promoter known in the artto promote transcription of operatively linked genes. The choice ofheterologous promoter will depend, for example, on whether it is desiredto turn on or off gene expression (in which case a regulated promoterwould be chosen), the desired level of gene expression (determining thechoice of strong or weak promoter), and the type of host cell or invitro transcription system (e.g. mammalian, bacterial, yeast,Drosophila, C. elegans, etc.). The promoter can alternatively be a STIMpromoter, such as a rat, hamster or other mammalian STIM1 promoter.STIM1 promoter sequences can be identified by those skilled in the artas regions within about 1000 nucleotides, such as within about 500, 200,100 or 50 nucleotides, of the transcriptional start of STIM1. Nucleicacid molecules operatively linked to a promoter of gene expression canbe used, for example, to express rodent STIM1, domains therefrom, andpolypeptides with substitutions thereto, in a desired host cell or invitro transcription-translation system. Methods for operatively linkinga nucleic acid to a promoter are well known in the art and include, forexample, cloning the nucleic acid into a vector containing the desiredpromoter, or appending the promoter to a nucleic acid sequence usingPCR.

Also provided are vectors containing the nucleic acid molecules. Thetype of vector will depend on the intended application. For example, inorder to amplify, subclone or genetically engineer a nucleic acidmolecule, a suitable cloning vector compatible with the host cell can beused, which may not have the capability to express the inserted nucleicacid molecules. In order to express mRNA or encoded polypeptides, thenucleic acid molecules can further be operatively linked to a promoterof gene expression, as described above, which may be a component of thevector or present in the inserted nucleic acid molecule. Factorsinvolved in ensuring compatibility between various host cells andvectors are well known in the art.

Further provided are cells containing nucleic acid molecules providedherein. Isolated nucleic acid molecule is generally contained within avector, which can be maintained episomally, or incorporated into thehost cell genome. Cells can be used, for example, for molecular biologyapplications such as to amplify, subclone or genetically engineer anucleic acid molecule. For such applications, bacterial cells, such aslaboratory strains of E. coli are useful. The cells also advantageouslycan be used to express the encoded polypeptide for screeningapplications, or to purify the encoded polypeptide. For suchapplications bacterial cells, insect cells (e.g. Drosophila), yeastcells (e.g. S. cerevisiae, S. pombe, or Pichia pastoris), and vertebratecells (e.g. mammalian primary cells and established cell lines; andamphibian cells, such as Xenopus embryos and oocytes), are useful.

Also provided are isolated oligonucleotides that contain at least 17contiguous nucleotides of SEQ ID NO:51 (or its complement) or SEQ IDNO:95 (or its complement), or SEQ ID NO:97 (or its complement),including at least one position selected from positions 15, 63, 69, 84,103, 108, 112, 120, 150, 183, 201, 207, 210, 213, 240, 300, 303, 312,330, 357, 402, 441, 474, 570, 621, 660, 697, 738, 783, 795, 861, 873,874, 895, 951, 1051, 1062, 1107, 1200, 1224, 1228, 1278, 1299, 1392,1395, 1452, 1580, 1652, 1654, 1675, 1747, 1749, 1173, 1854, 1855, 1881,1884, 1888, 1896, 2001 and 2025 of SEQ ID NO:51 or a position in SEQ IDNOS:95 or 97 that corresponds to the listed positions. The recitedpositions are those where the nucleotide of SEQ ID NO:51 differs fromthe corresponding position in SEQ ID NOS:2 and 82. One of skill in theart can use nucleotide alignment of one or more sequences with SEQ IDNO:51 to determine corresponding positions.

An oligonucleotide can contain the rodent reference, rat or hamsterSTIM1-specific nucleotide at the 5′ or 3′ end of the contiguousnucleotides of SEQ ID NO:51, or at any position within the contiguousnucleotides of SEQ ID NO:51. An oligonucleotide can contain severalrodent reference STIM1-specific nucleotide positions. For example, itcan include, among the at least 17 contiguous nucleotides of SEQ IDNO:51, nucleotides 63-69, or 103-120, or 201-213, or 300-312, or861-874, or 1224-1228, or 1392-1395, or 1652-1654, or 1747-1749, or1854-1855, or 1881-1896.

Also provided are fragments thereof or oligonucleotides that can be usedas probes or primers and that contain at least about 10, 14, 16nucleotides, generally less than 1000 or less than or equal to 100; orcontain at least about 30 nucleotides (or the complement thereof) orcontain oligonucleotides that hybridize along their full length or alongat least about 70%, 80% or 90% of the full length to any such fragmentsor oligonucleotides. The length of the fragments is a function of thepurpose for which they are used and/or the complexity of the genome ofinterest. Generally probes and primers contain less than about 500, 150,100 nucleotides.

In particular, oligonucleotides that include one or more nucleotidesthat are specific to rodent reference STIM1 nucleic acid molecules canspecifically hybridize to endogenous rat STIM1 DNA or mRNA, while notspecifically hybridizing to STIM1 nucleic acid molecules of otherspecies. Such oligonucleotides can be used, for example, as probes orprimers to determine the presence or amount of a rat STIM1-encodingnucleic acid molecule in a sample. Such oligonucleotides can also beprepared as inhibitory oligonucleotide molecules, such as RNAinterference, antisense or catalytic molecules, and used to specificallyreduce expression of a STIM1 nucleic acid molecule in vitro or in vivo.

It will be understood that 5′ or 3′ to the specified number ofnucleotides of rat STIM1, a nucleic acid molecule or oligonucleotide canoptionally contain heterologous nucleotide sequences, such as, but notlimited to, linkers or restriction sites useful in cloning applications;regulatory sequences useful in gene expression; sequences encodingepitope tags or fusion proteins useful in protein purification andanalysis.

The isolated nucleic acid molecules, including molecules encoding rodentreference STIM1, domains therefrom, and oligonucleotides, can beprepared by methods known in the art (see, for example, Sambrook andRussell (2000) “Molecular Cloning: A Laboratory Manual” Cold SpringHarbor Laboratory Press; Ausubel et al. (eds.) (current edition)“Current Protocols in Molecular Biology” John Wiley & Sons.

An exemplary method for preparing an isolated rodent reference STIM1nucleic acid molecule or oligonucleotide involves amplification of thenucleic acid molecule using STIM1-specific primers and the polymerasechain reaction (PCR). Using PCR, a rat STIM1 nucleic acid moleculehaving any desired boundaries can be amplified exponentially startingfrom only a few DNA or RNA molecules, such as from a single cell.

Alternatively, an isolated rodent reference STIM1 nucleic acid moleculeor oligonucleotide can be prepared by screening a library, such as acDNA library or expression library, with a detectably labeled rodentreference STIM1 nucleic acid molecule or antibody. Libraries arecommercially available or can be produced from cells of interest. Thelibrary clones identified as containing rodent reference STIM1 nucleicacid molecules can be isolated, subcloned and/or sequenced by methodsknown in the art.

Furthermore, an isolated rodent reference STIM1 nucleic acid molecule oroligonucleotide can be prepared by chemical or recombinant synthesis.For example, a single stranded nucleic acid molecule can be chemicallysynthesized in one piece, or in several pieces, by automated synthesismethods. The complementary strand can likewise be synthesized in one ormore pieces, and a double-stranded molecule made by annealing thecomplementary strands. Direct synthesis is particularly advantageous forproducing oligonucleotides, and also for producing nucleic acidmolecules containing modified nucleotides or linkages. A rodentreference STIM1 RNA (including an inhibitory RNA) can also be preparedrecombinantly by in vitro or in vivo transcription of a template DNAmolecule.

It will be understood that rodent reference STIM1 nucleic acid moleculesand oligonucleotides provide herein do not include, for example,identical sequences, including Expressed Sequence Tags (ESTs), SequenceTagged Sites (STSs), genomic fragments and other such fragments, thatmay have been previously deposited in public databases such as the NCBInr, dbest, dbsts, gss, pat and htgs databases (see, e.g.,www.ncbi.nlm.nih.gov/blast/). In particular, specifically excluded fromthe nucleic acid molecules provided herein are nucleic acid moleculehaving the exact, specific and complete nucleic acid molecule sequencecorresponding to the accession numbers that follow:gi|11632030|gb|BF524063.1|BF524063; gi|14950288|gb|BI291079.1|BI291079;gi|4280511|gb|AA996745.1|AA996745; gi|14947181|gb|BI289522.1|BI289522;gi|977818|gb|H32401.1|H32401; and gi|5209892|gb|AI763957.1|AI763957.

Vectors containing the nucleic acids encoding rodent reference STIM1 andcells containing the vectors are provided. Nucleic acid molecules,promoters, vectors and cells suitable for recombinantly producingpolypeptides and peptides having any desired boundaries, have beendescribed above. Rodent reference STIM1 polypeptides and domainstherefrom can also be prepared by biochemical procedures. A rat orhamster STIM1 polypeptide can be isolated from tissues or cells thatnaturally express the polypeptide, or from recombinant cells ortransgenic animals that express the polypeptide, by biochemicalprocedures routinely used in the art, including fractionation,chromatography, electrophoresis and affinity methods. Proteinpurification procedures are described, for example, in Rosenberg, I. M.(1996) “Protein Analysis and Purification: Benchtop Techniques” SpringerVerlag; and Scopes, R. K. (1994) “Protein Purification: Principles andPractice” Springer Verlag. For example, a rodent reference STIM1polypeptide can be isolated by immunoaffinity methods using theantibodies described herein.

b. Polypeptides

Also provided are mature rodent reference STIM1 polypeptides, domains ofrodent STIM1, and polypeptides with substitutions relative to rodentreference STIM1 or its domains, which retain at least one biologicalactivity of STIM1 or the domain. Rodent reference STIM1 polypeptidedomain boundaries, types of substitutions and biological activities havebeen described above with to nucleic acid molecules encoding suchpolypeptides. The polypeptides can be used, for example, in screeningapplications described herein, and to produce rodent reference STIM1antibodies. Isolated peptides provided herein as well as rodentreference STIM1 domain polypeptides, further can be used as agents thatblock interactions between STIM1 and its oligomeric partners.

An exemplary rat STIM polypeptide is set forth in SEQ ID NO. 98. Anexemplary hamster STIM polypeptide is set forth in SEQ ID NO. 96. Anexemplary reference STIM polypeptide is set forth in SEQ ID NO. 52. Asnoted also contemplated are rodent reference STIM polypeptides that havea specified homology and/or retain a biological activity. Included amongsuch variants are those encoded by the nucleic acid molecules describedabove. Variants include, but are not limited to, a polypeptide encodedby the sequence of nucleotides set forth in SEQ ID NOs. 51, 95 and 97; apolypeptide encoded by a sequence of nucleotides that hybridizes underconditions of low, moderate or high stringency to the sequence ofnucleotides set forth in SEQ ID NOs. 51, 95 and 97; a polypeptide thatcomprises the sequence of amino acids set forth in SEQ ID NO. 51, 96 or98; a polypeptide that comprises a sequence of amino acids having atleast about 60%, 70%, 80%, 90% or about 95% or more sequence identitywith the sequence of amino acids set forth in SEQ ID NO. 52, 96 or 98;and/or a polypeptide encoded by a splice variant of a sequence ofnucleotides that encodes a rodent reference STIM1 polypeptide. SEQ IDNO:52 sets forth the amino acid sequence of a full-length referenceSTIM1, as encoded by SEQ ID NO:51. Reference STIM1 differs from humanSTIM1 (SEQ ID NOS:4 and 83) and mouse STIM1 (SEQ ID NOS:10 and 85) atamino acid positions 21, 35, 38, 292, 527, 551, 552, 583 and 619. Theexemplified reference STIM1 differs from a human STIM1 polypeptides at atotal of 25 positions, and from a mouse STIM1 at a total of 17positions. Rat and hamster STIM1 differ from human STIM1 (SEQ ID NOS:4and 83) and mouse STIM1 (SEQ ID NOS:10 and 85) at multiple amino acidpositions, which can be determined by an alignment of the sequences.

Domains of hamster, rat and reference STIM1 can be determined by analignment with the relevant domains of human and mouse STIM1. Suchdomains include, for example, the extracellular, transmembrane,cytoplasmic, SAM, coiled-coil, Glu-rich, Pro-Ser-rich,ezrin/radixin/moesin (ERM), diacylglycerol kinase accessory domain(DAGKa) and Lys-rich domains.

In the exemplary reference STIM polypeptide set forth in SEQ ID NO: 52,amino acids 1-22 of SEQ ID NO:52 correspond to the signal peptidedomain, whereas amino acids 23-685 correspond to the mature referenceSTIM1 polypeptide. Amino acids 23-213 correspond to the extracellulardomain, amino acids 214-234 correspond to the transmembrane domain, andamino acids 235-685 correspond to the cytoplasmic domain of referenceSTIM1.

Within the extracellular domain, amino acids 132-200 correspond to a SAM(sterile alpha motif) domain. Because SAM domains in other proteins areinvolved in binding to SH2-containing polypeptides and inhomodimerization, it is contemplated herein that the SAM domain ofrodent reference STIM1 may mediate oligomerization with STIM1, STIM2and/or other polypeptides.

Within the cytoplasmic domain, amino acids 238-343 and 362-390correspond to coiled-coil domains. Amino acids 270-336 of the firstcoiled-coiled domain are rich in glutamic acid residues, and thus thisregion is designated as a Glu-rich domain. Because the coiled-coil motifis involved in oligomerization in many other proteins, it iscontemplated herein that either or both of the coiled-coiled domains ofrodent reference STIM1 (or the region corresponding from amino acids238-390 of reference STIM1), or the Glu-rich domain within its firstcoiled-coil domain, may mediate oligomerization with STIM1, STIM2 and/orother polypeptides.

Also within the cytoplasmic domain, amino acids 600-629 correspond to aproline-serine (Pro-Ser) rich domain. Because proline-rich andserine-rich domains are involved in oligomerization in many otherproteins, it is contemplated herein that the Pro-Ser rich domain ofrodent reference STIM1 may mediate oligomerization with STIM1, STIM2and/or other polypeptides.

Additional domains within the cytoplasmic domain of reference STIM1include an ezrin/radixin/moesin family “ERM” domain (amino acids253-424), a diacylglycerol kinase accessory domain “DAGKa” domain (aminoacids 422-484), and a lysine-rich domain (amino acids 672-685).

Also provided are isolated peptides that contain contiguous amino acidsof SEQ ID NO:52 that include a position selected from among positions21, 35, 38, 292, 527, 551, 552, 583 or 619 of SEQ ID NO:52 (orcorresponding positions). The recited positions are those where theamino acid residue in reference STIM1 differs from the correspondingresidue in a human and mouse STIM1.

A polypeptide provided herein can contain the rodent referenceSTIM1-specific amino acid at the N- or C-terminus of the contiguousamino acids of SEQ ID NO:52, or at any position within the contiguousamino acids of SEQ ID NO:52. It can contain several rodent referenceSTIM1-specific amino acid positions. For example, a polypeptide caninclude, among the at least 8 contiguous amino acids of SEQ ID NO:52,amino acids 35-38, or amino acids 551 and 552.

The polypeptides can be used, for example, in inducing and/or purifyingSTIM1 antibodies, including antibodies that specifically bind rodentreference STIM1 and not human or mouse STIM1. Rat, hamster and referenceSTIM1 peptides that are likely to be immunogenic, and thus useful forsuch applications, can be predicted using methods and algorithms knownin the art and described, for example, in Irnaten et al. (1998) ProteinEng. 11:949-955, and Savoie et al. (1999) Pac. Symp. Biocomput.1999:82-189. Immunogenicity can be tested or confirmed by methods knownin the art, such as by delayed-type hypersensitivity response assays inan animal sensitized to a STIM1 polypeptide, or by direct or competitiveELISA assays.

The number of substitutions to an encoded rodent reference STIM1polypeptide or domain therefrom that retains at least one biologicalactivity of the reference polypeptide or domain can be 1, 2, 3, 4 ormore, including any unit increment up to the total number of amino aciddifferences minus one between the rat and mouse or human sequence of thedomain of interest.

Rodent reference STIM1 polypeptide domains also can be produced byenzymatic or chemical cleavage of a longer rodent reference STIM1polypeptide. Methods for enzymatic and chemical cleavage and forpurification of the resultant fragments are well known in the art.Furthermore, rodent reference STIM1 domains and peptides can be producedby chemical synthesis methods known in the art (e.g. Grant, G. A. (ed)“Synthetic Peptides: A User's Guide” 2nd ed. Oxford University Press).

Biological activities of other STIM1 species (e.g. human, mouse andDrosophila), and methods of assessing such biological activities invitro or in vivo, are known in the art and/or described elsewhere hereinin detail. Because of the high degree of similarity between STIM1polypeptides across species, it will be appreciated that biologicalactivities of STIM1 from other species will be identical orsubstantially equivalent to the rat STIM1 polypeptides. It will also beappreciated that many of the biological activities of mature rodentreference STIM1, including the ability to oligomerize, will be exhibitedby the domains described herein, either alone or when expressed aschimeric proteins.

Assays that can be performed to confirm that a polypeptide withsubstitutions relative to mature rodent reference STIM1 (or a domaintherefrom) retains at least one biological activity of STIM1 (or of adomain therefrom) include, for example, assays that detect in vitro orin vivo homooligomerization; assays that detect in vitro or in vivoheterooligomerization with other polypeptides (e.g. STIM2); assays thatdetect binding to pre-B cells or differentiated B lymphocytes; assaysthat detect augmentation of IL-7-dependent proliferation followingexpression in pre-B cells; assays that detect modulation of cellmorphology following expression in 293T cells; assays that detectsuppression of cell growth following expression in tumor cells; andassays that detect modulation of intracellular calcium signaling.

c. Antibodies

Also provided are antibodies that specifically bind to isolated rat,hamster or reference STIM1 peptides, and antibodies that specificallybind to rat, hamster or reference STIM1 but not to either mouse STIM1 orhuman STIM1 or bind with substantially (at least 2, 5, 10-fold) lessaffinity. These antibodies have a variety of applications, such as, forexample, to detect rat and hamster STIM1 polypeptides expressed intissues or cells. Such antibodies are also used for purifying rodentreference STIM1 polypeptides by immunoaffinity methods. Furthermore,such antibodies can be used to interfere with the biological activity ofSTIM1 polypeptides or to deliver moieties to cells or tissues expressingSTIM1 polypeptides.

Methods of preparing and isolating antibodies, including polyclonal andmonoclonal antibodies and fragments therefrom, using rodent referenceSTIM1 polypeptides and peptides, as described above, are well known inthe art. Methods of preparing and isolating non-natural antibodies arealso well known in the art. For example, non-natural antibodies can beconstructed using solid phase peptide synthesis or can be producedrecombinantly, using nucleotide and amino acid sequence information ofthe antigen binding sites of antibodies that specifically bind thetarget polypeptide. Non-natural antibodies can also be obtained byscreening combinatorial libraries containing of variable heavy chainsand variable light chains, or of antigen-binding portions thereof.Methods of preparing, isolating and using polyclonal, monoclonal andnon-natural antibodies are reviewed, for example, in Kontermann andDubel, eds. (2001) “Antibody Engineering” Springer Verlag; Howard andBethell, eds. (2001) “Basic Methods in Antibody Production andCharacterization” CRC Press; and O'Brien and Aitkin, eds. (2001)“Antibody Phage Display” Humana Press.

D. Methods for Screening Agents and Molecules

Provided herein are methods of screening for or identify agents andmolecules that modulate intracellular calcium. The methods includescreening for or identifying a test agent that modulate intracellularcalcium. The methods also include identifying new molecules, e.g.,proteins and/or nucleic acids encoding new proteins, that werepreviously unknown, and for identifying known molecules (e.g., proteins)as being involved in intracellular calcium modulation. The test agentsand identified molecules can be used in the methods provided herein forscreening for agents for the treatment of a disease or disorder andadditionally in methods of treating diseases and disorders. The testagents and identified molecules can also be used to in methods such asprovided herein for modulating intracellular calcium.

1. Methods of Screening for or Identifying an Agent that ModulatesIntracellular Calcium

Methods of screening for or identifying agents that modulateintracellular calcium are provided herein. The methods are based indirectly or indirectly monitoring of or assessing the effect of testagents on intracellular calcium and/or in assessing the interaction of atest agent with, or the effect of a test agent on, a protein involved inmodulating intracellular calcium. In particular embodiments, the methodsinvolve monitoring of or assessing store-operated calcium entry, calciumlevels of (and/or movement of ions into, out of or within) intracellularorganelles or calcium stores (e.g., the endoplasmic reticulum),cytosolic calcium buffering and/or resting cytosolic calcium levels. Theeffect(s) of test agents on intracellular calcium can be assessed in avariety of ways including, but not limited to, evaluation of calcium orother ion (particularly cation) levels, movement of calcium or otherions particularly cations), fluctuations in calcium or other ion(particularly cation) levels, kinetics of calcium or other ionparticularly cation) fluxes and/or transport of calcium or other ion(particularly cation) through a membrane. The effect(s) of a test agentcan also be assessed by assays, such as described herein, to monitor acalcium-entry mediated event.

In one embodiment, the methods can be conducted using a particular testagent: one that binds to, interacts with and/or modulates interactions,activities, levels or any physical, structural or other property of aprotein involved in modulating intracellular calcium. In thisembodiment, the method includes monitoring or assessing the effects ofsuch a particular test agent on intracellular calcium (and, inparticular embodiments, the effects on store-operated calcium entry,calcium levels in or movement of ions (such as calcium) into, out of orwithin an intracellular organelle (or calcium store), cytosolic calciumbuffering and/or resting cytosolic calcium levels and/or effects on acalcium-entry mediated event). The method can optionally include a stepof identifying an agent that can bind to, interact with and/or modulateinteractions, activities, levels or any physical, structural or otherproperty of a protein involved in modulating intracellular calcium. Thisoptional step can be performed prior to or concurrently with the step ofmonitoring or assessing the effects of the agent on intracellularcalcium.

In another embodiment, the methods can be performed using a test agentthat modulates intracellular calcium and assessing the interaction ofthe test agent with, or the effect of the test agent on, a proteininvolved in modulating intracellular calcium. In particular embodiments,the binding of test agent with a protein involved in modulatingintracellular calcium can be assessed. In other embodiments, the effectof a test agent on interactions, activities, levels or any physical,structural or other property of a protein involved in modulatingintracellular calcium can be assessed. The method can optionally includea step of identifying a test agent that modulates intracellular calciumprior to (or concurrently with) the step of assessing the interaction ofthe test agent with, or the effect of the test agent on, a proteininvolved in modulating intracellular calcium.

In another embodiment, the methods can be performed by contacting anytest agent with (1) one or more proteins involved in modulatingintracellular calcium, e.g., a component of an ion transport proteincomplex or a modulatory protein, and/or (2) a cell, or portion thereof,e.g., a membrane, containing one or more proteins involved in modulatingintracellular calcium, and/or nucleic acid (e.g. a gene or codingsequence such as cDNA or RNA), or portion(s) thereof, encoding suchproteins. The effect of the test agent on intracellular calcium (and, inparticular embodiments, the effect on store-operated calcium entry,calcium levels in or movement of ions (such as calcium) into, out of orwithin an intracellular organelle (or calcium store), cytosolic calciumbuffering and/or resting cytosolic calcium levels) is monitored orassessed. In this embodiment of the methods, the test agent can be anyagent, and is not necessarily one that is known to modulate (or has beenidentified as one that modulates) intracellular calcium, or that bindsto, interacts with and/or modulates interactions, activities, levels orany physical, structural or other property of a protein involved inmodulating intracellular calcium.

In particular embodiments of any of the methods of screening for oridentifying an agent that modulates intracellular calcium providedherein, the protein(s) involved in modulating intracellular calcium canbe a protein that is involved in, participates in and/or provides forstore-operated calcium entry, cytosolic calcium buffering, maintenanceof resting cytosolic calcium levels and/or modulation of calcium levelsin, or movement of cations into, out of or within an intracellularorganelle or calcium store, such as, for example, the endoplasmicreticulum. In particular embodiments, the protein involved in modulatingintracellular calcium is an ion transport protein, such as, for example,an ion transport protein that is involved in, participates in and/orprovides for store-operated calcium entry or movement of calcium into,out of or within the endoplasmic reticulum or other calcium store. Inone embodiment, the protein involved in modulating intracellular calciumis a component of a store-operated calcium entry channel complex (e.g.,a multimeric complex containing multiple subunits of the same and/ordifferent proteins). In another embodiment the protein involved inmodulating intracellular calcium is a modulatory protein. A proteininvolved in modulating intracellular calcium (and/or nucleic acid, orportion(s) thereof, encoding a protein involved in modulatingintracellular calcium) may be contained in a cell or portion thereof,such as, for example, a cell membrane (e.g., plasma membrane or anintracellular membrane). The methods may be performed in particularembodiments under conditions that permit specific evaluation ofstore-operated ion flux or movement, resting cytosolic calcium levels,cytoplasmic calcium buffering and/or cation levels in, or movement into,out of or within an intracellular organelle or calcium store, such as,for example, the endoplasmic reticulum.

A protein involved in modulating intracellular calcium that is used inthe methods (or on which a method is based) can be a full-length orcomplete protein (e.g., a protein that contains the complete amino acidsequence encoded by a gene, cDNA or RNA or a complete amino acidsequence that lacks sequences that are removed during processing of theprotein, including removal of a signal sequence, such as may occur intransport of a protein to a particular cellular location, or processingto remove a pre- and/or pro-sequence of a protein) or a portion of acomplete protein. In embodiments of the methods that involve assessing afunctional activity of the protein, the protein used in the method canbe a portion of a full-length protein that is associated with, orexhibits or is sufficient for producing the functional activity, e.g.,an intracellular calcium-modulating activity. In embodiments of themethods that involve assessing a property of the protein that is notnecessarily the intracellular calcium-modulating activity of theprotein, the protein used in the method can be a portion of afull-length protein that is associated with the particular propertybeing assessed, e.g., binding properties, and can be, for example, aparticular domain of the protein. Similarly, when a nucleic acid, orportion(s) thereof, encoding a protein involved in modulatingintracellular calcium is used in the methods, the nucleic acid can be acomplete gene, e.g., including transcriptional regulatory sequences, acomplete protein coding sequence, e.g., cDNA or RNA, or portion(s) ofthese (e.g., a portion encoding a functional domain of a protein).

a. Proteins (and/or Nucleic Acids Encoding Proteins) Involved inModulating Intracellular Calcium

The methods provided herein for screening for or identifying agents thatmodulate intracellular calcium are related to proteins (and/or nucleicacids, or portions thereof, that encode proteins) that are involved inmodulating intracellular calcium. Some embodiments of the methodsinvolve monitoring or assessing the effect on intracellular calcium of atest agent that binds to, interacts with and/or in some way modulatessuch a protein. Other embodiments involve the actual use of suchproteins. For example, one embodiment involves assessing the interactionof a particular test agent with, or the effect of a particular testagent on, such a protein. Another embodiment involves contacting a testagent with (1) one or more proteins involved in modulating intracellularcalcium, e.g., a component of an ion transport protein complex or amodulatory protein, and/or (2) a cell, or portion thereof, e.g., amembrane, containing one or more such proteins, and/or nucleic acid(e.g., a gene or coding sequence such as cDNA or RNA), or portion(s)thereof, encoding such proteins.

Proteins involved in modulating intracellular calcium can be, forexample, ion transport proteins, a component of an ion transport proteincomplex, calcium-binding proteins, modulatory proteins, receptors andregulatory proteins that regulate ion transport proteins, receptors orcalcium-binding proteins. Proteins (or nucleic acids, or portion(s)thereof, encoding proteins) that can be used in the methods of screeningfor or identifying agents that modulate intracellular calcium providedherein (or proteins on which the methods are based) include proteinsthat are homologous to a protein encoded by a Drosophila or mammalian(e.g. human or rodent, such as rat, hamster or mouse) gene that, whenaltered in its expression in a cell, results in altered intracellularcalcium. An alteration in intracellular calcium can be any alteration incalcium level, movement, location, or other calcium alteration, in acell. An alteration of intracellular calcium can be any change inintracellular calcium compared to a control cell (e.g., a Drosophila ormammalian cell that does not have altered expression of the gene).

In particular embodiments, the protein can be one that is homologous toa protein encoded by a Drosophila or mammalian (e.g., human or rodent,such as rat or mouse) gene that when altered in its expression in a cell(e.g., a Drosophila cell or mammalian, such as human or rodent, cell),results in altered store-operated calcium entry into the cell, alteredcalcium levels in or altered calcium movement into, within or out of anintracellular organelle or calcium store, altered cytosolic calciumbuffering and/or altered basal or resting cytosolic calcium levels. Analteration of store-operated calcium entry or of movement into, out ofor within an intracellular organelle or calcium store (e.g., endoplasmicreticulum) can be a complete or nearly complete elimination of theactivity, a reduction of the activity, an alteration in properties orcharacteristics of the activity (e.g., current properties orsensitivities) or an increase in the activity, e.g., relative to theactivity in a control cell (e.g., a Drosophila or mammalian cell), thathas not been altered in its store-operated ion flux activity.

An alteration in the calcium level within an intracellular organelle oran alteration of resting cytosolic calcium levels can be, for example, areduction, depletion, elimination of, or increase in calcium levels,e.g., relative to the levels in a control cell (e.g., a Drosophila ormammalian cell) that has not been altered in its intracellular organelleor basal cytosolic calcium levels. An alteration in the calcium level ofan organelle or calcium store or an alteration in basal cytosoliccalcium level can be effected in a number of ways, including, forexample, by alterations in calcium movement across a cellular membrane(e.g., plasma membrane or membrane of an intracellular organelle orcalcium store).

An alteration in cytosolic calcium buffering can be, for example, acomplete or nearly complete elimination of the activity, a reduction ofthe activity, an alteration in properties or characteristics of theactivity (e.g., rates, kinetics or timing) or an increase in theactivity, e.g., relative to the activity in a control cell (e.g., aDrosophila or mammalian cell) that has not been altered with respect toits calcium buffering activity. Thus, for example, an alteration incalcium buffering can be a reduction or increase in the rate at whichcytosolic calcium levels return to basal levels after activation ofcalcium influx into the cytoplasm. The alteration can be an overall timecourse of cytosolic calcium level adjustment that differs from that in acontrol cell. In another example, an alteration in calcium buffering canbe a delay in onset of the adjustment of cytosolic calcium levels toreturn to basal levels after activation of calcium influx into thecytoplasm. In another example, an alteration in calcium buffering can bean adjustment in cytosolic calcium levels after activation of calciuminflux that does not result in a return of calcium levels to a basallevel, but instead to a level that is higher or lower than the basallevel. In yet a further example, an alteration in calcium buffering canbe a complete or near complete absence of an adjustment in cytosoliccalcium levels following activation of calcium influx into thecytoplasm.

Similarly, an alteration in gene expression may be complete or nearlycomplete elimination of the expression of a gene, a reduction in theexpression of a gene, an increase in the expression of a gene, or analteration in the protein encoded by the gene (such as truncation orother alteration that effectively renders the protein nonfunctional orprovides for aberrant functioning of the protein), e.g., relative to theexpression of the gene in a cell that has not been altered in itsexpression of the gene.

In a particular embodiment of the methods of screening for oridentifying an agent that modulates intracellular calcium, the proteinused in the method (and/or protein encoded by a nucleic acid, orportion(s) thereof, used in the method) can be one that is homologous toa protein encoded by a Drosophila or mammalian (e.g., human or rodentsuch as rat, hamster or mouse) gene that, when altered in its expressionin (e.g., a Drosophila or mammalian cell), results in alterations in oneor more, or all, of the following: (1) store-operated calcium entry intothe cell, (2) calcium levels in an intracellular organelle or calciumstore, (3) calcium movement into, within or out of an intracellularorganelle or calcium store and (4) cytosolic calcium buffering.

Particular proteins that are homologous to a protein encoded by aDrosophila or mammalian gene that, when altered in its expression,results in altered intracellular calcium include proteins that areinvolved in, participate in and/or provide for the movement of calcium.Such proteins may be relatively specific for calcium ion transport. Forexample, the protein can be an ion transport protein, or a component ofan ion transport protein complex, that is involved in, participates inand/or provides for store-operated calcium entry.

In one embodiment of the methods of screening for or identifying agentsthat modulate intracellular calcium provided herein, a protein used inthe method, or on which the method is based, is involved in modulatingintracellular calcium (and, in particular embodiments, is involved in,participates in and/or provides for store-operated calcium entry,movement of calcium into, out of or within an intracellular organelle,modulation of intracellular organelle calcium level, and/or cytosoliccalcium buffering) and is homologous to a protein encoded by the codingsequence of Drosophila gene CG9126 (GenBank Accession no. NM_(—)078633,gi22832319 and AF328906; see also SEQ ID NO:1 for a coding sequence andSEQ ID NO: 2 and GenBank Accession no. NP_(—)523357, AAF48542, AAK82338and P83094 for amino acid sequence) and/or a mammalian stromalinteracting molecule (STIM) protein (see, e.g., SEQ ID NO:90 for amammalian STIM1 consensus amino acid sequence), such as, for example,human STIM1 (GenBank protein Accession nos. Q13586, NP_(—)003147,AAC51627 and nucleotide Accession nos. NM_(—)003156, gi2264345,gi2264346; see also SEQ ID NOS:3, 82 for nucleic acid coding sequencesand SEQ ID NOS: 4 and 83 for amino acid sequences), rat STIM 1 (see SEQID NO: 97 for a nucleic acid coding sequence and SEQ ID NO: 98 for anamino acid sequence, hamster STIM1 (see SEQ ID NO: 95 for a nucleic acidcoding sequence and SEQ ID NO: 96 for an amino acid sequence) andreference STIM1 (see SEQ ID NO: 51 for a nucleic acid coding sequenceand SEQ ID NO: 52 for an amino acid sequence). As described herein (seethe EXAMPLES), alteration, and, in particular, reduction, of theexpression of CG9126 in Drosophila S2 cells or of the expression of thegene encoding mammalian STIM1 proteins in mammalian cells (e.g., humanSH-SY5Y and HEK293 cells, rat basophilic leukemia (RBL-2H3) cells andChinese hamster ovary (CHO) cells) is associated with a reduction instore-operated calcium entry into the cells, a reduction in calciumlevels in the endoplasmic reticulum, a reduction in movement of calciumout of the endoplasmic reticulum and/or alteration of calcium bufferingfollowing activation of release of calcium from intracellular calciumstores. Accordingly, the protein encoded by CG9126, and mammalian (e.g.,human and rodent, such as rat) STIM proteins, as well as proteinshomologous with CG9126 and mammalian (e.g., human and rodent, such asrat and hamster) STIM proteins, are identified herein as being involvedin modulating intracellular calcium. In particular, such proteins areidentified herein as being involved in, participating in and/orproviding for store-operated calcium entry, modulation of calcium levelsin or movement of calcium into, out of, or within intracellular calciumstores (e.g., endoplasmic reticulum) and/or calcium buffering. Inparticular embodiments, a protein used in the method (or on which themethod is based) that is involved in modulating intracellular calciumand is homologous to a specified protein is an ion transport protein, acomponent of an ion transport protein complex, a modulatory protein, ora receptor.

A protein used in the methods of screening for or identifying an agentthat modulates intracellular calcium can be, for example, one that isinvolved in modulating intracellular calcium (and, in particularembodiments, is involved in, participates in and/or provides forstore-operated calcium entry, movement of calcium into, out of or withinan intracellular organelle, modulation of intracellular organellecalcium level, and/or cytosolic calcium buffering) and that has an aminoacid sequence that is at least about 20%, or at least about 25%, or atleast about 30%, or least about 35%, or at least about 39%, or at leastabout 40%, or at least about 45%, or at least about 47%, or at leastabout 50%, or at least about 55%, or at least about 60%, or at leastabout 65%, or at least about 70%, or at least about 75%, or at leastabout 80%, or at least about 85%, or at least about 90%, or at leastabout 95% or more homologous to an amino acid sequence of the proteinencoded by the coding sequence of Drosophila gene CG9126 and/or amammalian stromal interacting molecule (STIM) protein, e.g., human orrodent (such as rat or hamster) STIM1. The particular homology candepend on the particular protein, e.g., species, that is homologous tothe specified proteins and the extent of a specified protein to whichthe particular protein is homologous. In particular embodiments, theprotein is at least 45% or more homologous to the protein encoded by thecoding sequence of Drosophila gene CG9126 and/or a mammalian stromalinteracting molecule (STIM) protein, e.g., human or rodent (such as rator hamster) STIM1. Such exemplary proteins may be homologous to thespecified proteins over at least about 25%, or at least about 30%, or atleast about 35%, or at least about 40%, or at least about 45%, or atleast about 50%, or at least about 52%, or least about 55%, or at leastabout 60%, or at least about 65%, or at least about 70%, or at leastabout 75%, or at least about 80%, or at least about 84%, or at leastabout 85%, or at least about 90%, or at least about 95% or more of theprotein encoded by the coding sequence of a specified protein. Inparticular embodiments, the protein is homologous to the specifiedproteins over at least about 52% or more of a specified protein.

In particular embodiment of the methods for screening for or identifyingagents that modulate intracellular calcium, a protein used in the method(or proteins encoded by nucleic acids, or portion(s) thereof, used inthe method) is one that is involved in modulating intracellular calcium(and, in particular embodiments, is involved in, participates in and/orprovides for store-operated calcium entry, movement of calcium into, outof or within an intracellular organelle or calcium store, modulation ofintracellular organelle (or calcium store) calcium level, and/orcytosolic calcium buffering) and that has an amino acid sequence that isat least about 45% homologous over at least about 52% of a specifiedprotein.

Proteins homologous to the protein encoded by the coding sequence ofDrosophila gene CG9126 and/or a mammalian stromal interacting molecule(STIM) protein, e.g., human or rodent STIM1, include, but are notlimited to, the proteins listed in Table 3. TABLE 3 Examples of ProteinsHomology (Identity) Homology (Identity) PROTEIN(GenBank To ProteinEncoded Homology To Protein; Nucleic Acid By Drosophila Gene (Identity)To Reference Accession Numbers) CG9126 Human STIM1 STIM1 Protein EncodedBy 100% (100%) over 58% (39%) over 58% (39%) Drosophila Gene 100% of theprotein 70% of the over 70% of CG9126SEQ ID protein the protein NOS: 2,75-81 (NP_523357, AAF48542, AAK82338, P83094 and AAL39831; NM_078633,gi22832319, AF328906 and AY069686) Homo sapiens 58% (39%) over 84% 100%(100%) 97% (96%) STIM1SEQ ID NOS: 3, of the protein over 100% of theover 100% of 4, 82-84(Q13586, protein the protein NP_003147, AAC51627;NM_003156, U52456, gi2264346) Reference STIM1SEQ 58% (39%) over 85% 97%(96%) over 100% (100%) ID NOS: 51 and 52 of the protein 100% of the over100% of protein the protein Mus musculus 58% (39%) over 84% 97% (96%)over 98% (97%) STIM1SEQ ID NOS: 9, of the protein 100% of the over 100%of 10 and 85(NP_033313.1 protein the protein and P70302; NM_009287) Homosapiens unknown 58% (39%) over 84% 99% (99%) over 97% 96%) protein forof the protein 100% of the over 100% of MGC: 29566SEQ ID protein theprotein NOS: 49 and 50(AAH21300; BC021300) Mus musculus stromal 58%(39%) over 84% 97% (96%) over 98% (97%) cell proteinSEQ ID of theprotein 100% of the over 100% of NOS: 56 and protein the protein55(AAC52715; U47323) Homo sapiens 61% (41%) over 75% 66% (53%) over 67%(59%) KIAA1482 proteinSEQ of the protein 84% of the over 80% of ID NOS:59 and protein the protein 60(BAA96006; AB040915) Homo sapiens 61% (41%)over 75% 66% (53%) over 68% (53%) STIM2SEQ ID NOS: 5, of the protein 84%of the over 80% of 6, 86-88(AAK82337, protein the protein NP_065911,Q9P246; AF328905, NM_020860) Mus musculus 60% (41%) over 66% 64% (52%)over 65% (53%) STIM2SEQ ID NOS: of the protein 80% of the over 80% of11, 12, 67, protein the protein 68(XP_132038, AAK82339; XM_132038,AF328907) Rattus norvegicus 58% (40%) over 63% 71% (58%) over 72% (59%)protein similar to of the protein 63% of the over 61% of STIM2 SEQ IDNOS: protein the protein 72, 73(XP_223454; XM_223454) Homo sapiensprotein 58% (40%) over 53% 67% (56%) over 71% (60%) similar to STIM2SEQof the protein 59% of the over 52% of ID NOS: 61 and protein the protein62(AAH15659; BC015659) Anopheles gambiae str. 85% (73%) over 84% 59%(40%) over 60% (42%) PEST ebiP8103 SEQ ID of the protein 71% of the over68% of NOS: 6 and protein the protein 7(EAA06485; AAAB1008846) Musmusculus protein 58% (39%) over 84% 97% (96%) over 98% (97%) for MGC:13964SEQ ID of the protein 100% of the over 100% of NOS: 53 and proteinthe protein 54(AAH21644; BC021644) Homo sapiens 61% (41%) over 75% 66%(53%) over 67% (55%) hypothetical protein of the protein 84% of the over80% of SEQ ID NOS: 57 and protein the protein 58(CAB66512; AL136577)Caenorhabditis elegans 50% (31%) over 74% 52% (31%) over 52% (31%)Y55B1M.1.p protein of the protein 56% of the over 56% of SEQ ID NOS: 13and protein the protein 14(NP_497197; NM_064796) Caenorhabditis elegans51% (32%) over 67% 49% (29%) over 47% (28%) putative membrane of theprotein 68% of the over 74% of protein SEQ ID NOS: protein the protein73 and 74(NP_741073; NM_171065) Caenorhabditis elegans 50% (31%) over74% 52% (31%) over 52% (31%) hypothetical protein of the protein 56% ofthe over 56% of Y55B1BM.1aSEQ ID protein the protein NOS: 63 and64(AAF59596; AC024823) Caenorhabditis elegans 51% (32%) over 67% 49%(29%) over 47% (28%) hypothetical protein of the protein 68% of the over75% of Y55B1BM.1bSEQ ID protein the protein NOS: 65 and 66(AAL32257;AC024823)

In other embodiments, other proteins that may be used in the methodsprovided herein include, but are not limited to, proteins involved inintracellular calcium modulation that are substantially homologous tothe above-listed proteins.

These listed proteins and their amino acid sequences, and nucleic acidsencoding such proteins (including the listed nucleotide sequences),exemplify proteins and nucleic acids that are useful for methods,materials and systems provided herein. Naturally occurring andsynthesized alternative forms of these proteins such as allelic forms,isoforms, muteins, and mutated derivatives will have sequence changesthat do not abolish their operation in intracellular calcium modulationand are useful in embodiments. Mutations, polymorphisms and allelicforms of these proteins are known and are contemplated as embodiments.

Proteins used in the methods for screening for or identifying an agentthat modulates intracellular calcium include synthetic proteins,proteins endogenously expressed by a cell, and recombinant proteins. Theprotein can, for example, be isolated from a source containing theprotein or can be contained within a cell, virus or organism orportion(s) thereof, e.g., a membrane. Nucleic acids used in the methodsinclude synthetic nucleic acids, recombinant nucleic acids and nucleicacids isolated from a source containing the nucleic acid. A variety ofmethods, some of which are described herein, can be employed for proteinand nucleic acid synthesis and isolation, and recombinant expression ofnucleic acids and proteins. Many such methods are well known in the art.

b. Test Agents

Generally, agents tested in the methods of screening for or identifyingagents that modulate intracellular calcium provided herein can be of anyphysical type. Examples of agents include, but are not limited to,biomolecules, including, but not limited to, amino acids, peptides,polypeptides, peptiomimetics, nucleotides, nucleic acids (including DNA,cDNA, RNA, antisense RNA and any double- or single-stranded forms ofnucleic acids and derivatives and structural analogs thereof),polynucleotides, saccharides, fatty acids, steroids, carbohydrates,lipids, lipoproteins and glycoproteins. Such biomolecules can besubstantially purified, or can be present in a mixture, such as a cellextract or supernate. Test agents further include synthetic or naturalchemical compounds, such as simple or complex organic molecules,metal-containing compounds and inorganic ions. Also included arepharmacological compounds, which optionally can be subjected to directedor random chemical modifications, such as acylation, alkylation,esterification, amidation, etc., to produce structural analogs.

Test agents suitable for use in the methods can optionally be containedin compound libraries. Methods for producing compound libraries byrandom or directed synthesis of a wide variety of organic compounds andbiomolecules are known in the art, and include expression of randomizedoligonucleotides and oligopeptides. Methods of producing naturalcompounds in the form of bacterial, fungal, plant and animal extractsare also known in the art. Additionally, synthetically produced ornatural compounds and compound libraries can be readily modified throughconventional chemical, physical and biochemical means to producecombinatorial libraries. Compound libraries are also available fromcommercial sources.

c. Methods Based on Testing an Agent that Binds to, Interacts withand/or Modulates Interactions, Activities or Levels of a ProteinInvolved in Modulating Intracellular Calcium (and/or Nucleic Acid, orPortion(s) Thereof, Encoding such Proteins)

In one embodiment of the methods for screening for or identifying agentsthat modulate intracellular calcium provided herein, the test agent isone that binds to, interacts with and/or modulates interactions,activities, levels or any physical, structural or other property of aprotein (e.g., an ion transport protein, a component of an ion transportprotein complex, a modulatory protein, or a receptor) involved inmodulating intracellular calcium and/or portion(s) thereof. Thisembodiment of the methods includes a step of assessing or monitoring orassessing the effects of such a test agent on intracellular calcium(and, in particular embodiments, the effects on store-operated calciumentry, calcium levels in or movement of calcium into, out of or withinan intracellular organelle (or calcium store), cytosolic calciumbuffering and/or resting cytosolic calcium levels). Such particular testagents used in this embodiment include agents that are known to be orthat have already been identified as agents that can bind to, interactwith and/or modulate interactions, activities, levels or any physical,structural or other property of a protein involved in modulatingintracellular calcium. Optionally, this embodiment of the methods caninclude a step of identifying an agent that can bind to, interact withand/or modulate interactions, activities, levels or any physical,structural or other property of a protein involved in modulatingintracellular calcium.

The protein involved in modulating intracellular calcium that the testagent binds to, interacts with and/or modulates (e.g., the interactions,activities, levels or any physical, structural or other property of theprotein) can be a protein as provided herein and described above (andelsewhere herein). In particular embodiments, the protein is one that isinvolved in, participates in, and/or provides for store-operated calciumentry, movement of calcium into, out of or within an intracellularorganelle or calcium store, modulation of intracellular organelle (orcalcium store) calcium level, and/or cytosolic calcium buffering. Inparticular embodiments, the protein is one of the proteins (or issubstantially homologous to one of the proteins) listed in Table 3. Theprotein can be a STIM or STIM-like protein, including a STIM1, STIM2,DSTIM or CSTIM protein. In one embodiment of the methods, the protein isa STIM1 protein, for example, a mammalian STIM1 protein. In a particularembodiment, the effect of test agent on store-operated calcium entry ismonitored or assessed.

i. Identification of Particular Test Agents

Test agents suitable for use in this embodiment of the methods forscreening for or identifying agents that modulate intracellular calciumcan be identified in a number of ways using techniques described hereinand/or known in the art.

(a) Binding and Interaction Assays

A number of in vitro and cell-based binding assays are known in the artand can be modified as needed by one of skill in the art to identifyagents that bind to or interact with a protein involved in modulatingintracellular calcium and/or portion(s) thereof. Test agents that bindto a protein involved in modulating intracellular calcium includemolecules that physically interact with the protein and/or portion(s)thereof with relatively high affinity and selectivity. For example, anagent that binds to a protein involved in modulating intracellularcalcium can bind with a K_(d) of about 10⁻⁴ M or less, such as about10⁻⁶ M or less, including about 10⁻⁸ M or about 10⁻⁹ M or less. Incontrast, under the same conditions, the agent can bind a protein thatis not the particular protein involved in modulating intracellularcalcium with an affinity that is at least 10-fold lower, such as atleast 100-fold or 1000-fold lower.

In vitro methods for identifying molecules that bind to or interact witha protein involved in modulating intracellular calcium include bothdirect methods, e.g., in which binding between the agent and thepolypeptide is detected or measured, and competitive methods, in whichthe ability of an agent to displace binding between a bound molecule andthe polypeptide is detected or measured. For example, antibodies, suchas monoclonal antibodies, that specifically recognize a protein ofinterest or portion thereof can be used to compete for binding of theprotein or portion thereof.

Exemplary in vitro methods include co-purification assays (e.g., GSTpull-down assays, co-immunoprecipitation assay, chromatographic assays),phage display (see, e.g., Rodi et al. (2002) Curr. Opin. Chem. Biol.6:92-96), ribozyme display (Hanes and Pluckthun (1997) Proc. Natl. Acad.Sci. U.S.A. 13:4937-4942), and protein arrays (Cahill (2001) J. Immunol.Meth. 250:81-91). Antibodies that recognize a particular protein can beused to immunoprecipitate the protein and any molecule(s) bound to theprotein. Lambda phage expression libraries can also be screened, usingmethods known in the art, for proteins that bind to a protein ofinterest or portion thereof.

Detection of in vitro binding or interaction between an agent and aprotein involved in modulating intracellular calcium and/or portion(s)thereof can involve a variety of approaches, such as, for example,nuclear magnetic resonance (NMR) (Hadjuk et al. (1999) J. Med. Chem.42:2315-2317), mass spectroscopy (Siegel (2002) Curr. Top. Med. Chem.2:13-33), fluorescence spectroscopy (Winkler et al. (1999) Proc. Natl.Acad. Sci. U.S.A. 96:1375-01378), scintillation proximity assays (SPQ)(Fernandes (1998) Curr. Opin. Chem. Biol. 2:597-603), surface plasmonresonance assays (available commercially from BIACORE;/www.biacore.se/proteomics/), and others. Many of these methods areamenable to high-throughput screening of test agents.

The protein involved in modulating intracellular calcium, the agent, orboth, used in an in vitro binding or interaction assay can be in gasphase, in solution, in suspension, or attached to a solid support, asappropriate for the assay method. The protein, agent, or both can bedetectably labeled. Methods for preparing the protein or test agent in aform suitable for the particular assay are known in the art. Forexample, the protein, or a portion thereof, can be preparedsynthetically, isolated from a source of the protein, enzymatically orotherwise cleaved to yield desired protein or peptide fragments, orproduced by recombinant methods, such as by expression of a nucleic acidconstruct encoding the protein (or portion thereof) in a host cell(e.g., bacteria, yeast, or mammalian cell or other cells) and secretionor isolation therefrom.

Cell-based binding assays include, for example, yeast two-hybrid assays(see, e.g., U.S. Pat. Nos. 5,283,173, 5,468,614 and 5,667,973 and PCTApplication Publication Nos. WO01/25420 and WO02/079493); bacterialtwo-hybrid assays (Juong (2001) J. Cell Biochem. Suppl. 37:53-57), andothers. Such assays are particularly suitable for identifyingpolypeptides that interact with a protein involved in modulatingintracellular calcium. Two-hybrid assays are based on the modular natureof most transcription factors, which consist of separable DNA-bindingand activation domains. Briefly, such assays use two different DNAconstructs, one that codes for the polypeptide of interest (i.e., aprotein involved in modulating intracellular calcium or portion thereof)fused to a gene or nucleic acid encoding the DNA binding domain of aknown transcription factor. In the other construct, a DNA sequence, froma library of sequences, that encodes a potential test agent polypeptideis fused to a gene or nucleic acid that codes for the activation domainof the known transcription factor. If the polypeptide of interest andthe test polypeptide interact, the DNA-binding and activation domainsare brought into close proximity, which allows transcription of areporter gene that is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected, and cell colonies containing the functionaltranscription factor isolated. From these colonies, the nucleic acidmolecule encoding the test polypeptide that binds or interacts with theprotein involved in intracellular calcium modulation can be isolated.

Another cell-based assay for identification of molecules that interactwith a protein, such as a protein involved in modulating intracellularcalcium, is the tandem affinity purification (TAP) method (see, e.g.,Rigaut et al. (1999) Nature Biotech. 17:1030-1032; Puig et al. (2001)Methods 24:218-229). The method involves a two-step process for theaffinity purification of protein complexes from cells, e.g., yeast, whenexpressed at relatively normal levels under native conditions. Themethod employs a tag that is fused to the protein of interest (e.g., aprotein involved in modulating intracellular calcium), or portion(s)thereof, through recombinant expression of the fusion protein in orderto facilitate affinity purification using two different selectionmolecules. The tag can be a fusion of protein A IgG-binding domains (foruse in a first affinity purification by an IgG matrix) and acalmodulin-binding peptide (for use in a second affinity purification bycalmodulin-coated beads) separated by a TEV protease cleavage site (toeluate protein complexes bound to the IgG matrix). Thus, the protein ofinterest is expressed in cells as a fusion with the tag and thenextracts of the cells are mixed with the IgG matrix, bound complexes areeluted by cleavage with TEV protease, and the eluate is mixed withcalmodulin-coated beads in the presence of calcium. The complexes arereleased from the beads with EGTA. The purified complex can be used forprotein identification, functional or structural studies. Analyticalmethods that can be applied include analytical gel electrophoresis andmass spectrometry. The method can be used to identify a variety ofinteracting agents, including, for example, proteins, nucleic acids,lipids and peptides.

Assays that generally can be used for identifying test agents thatmodulate the binding or interaction of a protein of interest (e.g., aprotein involved in intracellular calcium modulation), or portion(s)thereof, with other molecules are also known in the art. In theseassays, a complex of the protein of interest and a binding/interactionpartner (or portion(s) thereof) is typically contacted with or exposedto a potential test agent and the binding or interaction of the complexis assessed (for example, for an effect of the potential test agent onthe binding, such as decreased, increased or other alterations in theinteraction) using standard analytical techniques as described hereinand known in the art. For example, one such technique involves measuringfluorescence resonance energy transfer (FRET) between an energy donormolecule linked to one binding partner and an energy acceptor moleculelinked to the other binding partner in the presence of an agent. Anagent that alters the interaction of the binding partners can bedetected as a difference (e.g., increase or fluorescent energy of adifferent wavelength) in the fluorescence of the sample. Suitable donorand acceptable molecules and conditions for conducting such assays areknown in the art (see, e.g., U.S. Pat. Nos. 6,348,322 and 4,868,103).FRET assays may also be used to identify molecules that interact with orbind to a protein of interest (e.g., a protein involved in modulatingintracellular calcium) or portion(s) thereof. Other methods that can beused to assess binding between binding partners include ELISA andradioligand binding assays.

As described above, a protein involved in modulating intracellularcalcium that is used in the methods for screening for or identifying anagent that modulates intracellular calcium (or a protein on which themethod is based) can be a full-length or complete protein or a portionof a complete protein. Portions of complete proteins include portionsthat are associated with, exhibit or that are sufficient for producing aparticular activity or function of the complete protein, and portionsthat are associated with a particular property or feature of thecomplete protein, such as a binding portion or a particular domain, orany random portion of the protein. Similarly, nucleic acids that encodeproteins that can be used in the methods (or on which a method is based)can encode a complete protein or portion(s) thereof.

In the embodiment of the methods that involve assessing the effects onintracellular calcium of an agent that binds to or interacts with aprotein involved in modulating intracellular calcium, the test agent canbe one that is identified, for example, using any suchbinding/interaction assay as described above (or known in the art) thatemploys a complete or full-length protein of interest or portion(s)thereof. Portions of proteins can be obtained using a variety of methods(e.g., synthetically, through recombinant production methods) describedherein and/or known in the art and can be random or selected for aparticular feature. Methods for identifying particular features ofproteins are known in the art. For example, particular domains (e.g.,protein interaction domains, calcium-binding domains, catalytic domains,phosphorylation domains and others) can be identified by comparing theprimary structure of a protein to known motifs and domains, particularlythrough computer-assisted comparisons utilizing protein motif databases(see, e.g., computer-assisted proteome analysis programs such as thatavailable through www.ebi.ak.uk/proteome, and searchable amino acidsequence pattern information available and searchable through otherdatabases, including InterPro (see www.ebi.ac.uk/interpro/) as well asSWISS-PROT, TrEMBL, PROSITE, PRINTS, Pfam and Prodom; see also thedomain identification and Conserved Domain Architecture Retrieval Tool(CDART) programs available through the National Center for BiotechnologyInformation (NCBI) Entrez Search and Retrieval system(www.ncbi.nlm.nih.gov)).

In a particular embodiment of these methods of screening for oridentifying an agent that modulates intracellular calcium, a proteinused in the method (or on which the method is based) is a STIM orSTIM-like protein (e.g., a STIM1 or STIM2 or DSTIM protein). The testagent can be one that binds to or interacts with a STIM or STIM-likeprotein or portion(s) thereof or that modulates the interaction orbinding of such a protein with a binding partner. Such agents can be anyagent known to bind to or interact with such proteins (or known tomodulate the binding or interaction of such proteins) or can be oneidentified using methods described herein and/or known in the art. Anyportion of a STIM or STIM-like protein can be used in methods ofidentifying an agent that binds to or interacts with the protein,including the complete protein, the mature protein (lacking signalsequence) and any portion thereof. Particular domains of STIM andSTIM-like proteins are described herein and/or can be identified usingmethods described herein and/or known in the art. For example, sterile amotif (SAM) and coiled-coil domains contained in N-terminal andC-terminal regions, respectively, of STIM proteins are domainsimplicated in protein-protein interactions and subunit oligomerization.Examples of nucleic acids encoding STIM proteins and portions thereofare also described herein or can be designed based on amino acidsequences such as those described herein.

In a particular embodiment of these methods, a test agent is one thatmodulates the interaction of two or more STIM or STIM-like proteins (orportion(s) thereof). For example, human STIM1 and STIM2 proteins formco-precipitable oligomeric associations in K562 cells (chronic myeloidleukemia cells) and in 293T cells transfected with nucleic acidsencoding human STIM1 and STIM2 proteins (Williams et al. (2001) Biochem.J. 357:673-685). In addition, STIM1 homotypic interactions occur inhuman 293T cells transfected with nucleic acid encoding human STIM1 andnucleic acid encoding a fusion protein containing the transmembrane andcytoplasmic (i.e., the C-terminal) region of human STIM1 (Williams etal. (2002) Biochim. Biophys. Acta 1596:131-137). Thus, STIM1-STIM1homotypic interactions are mediated via the C-terminal domain of theprotein. Methods of assaying the interaction of STIM proteins includemethods described by Williams et al. ((2001) Biochem. J. 357:673-685;and Biochim. Biophys. Acta 1596:131-137), including cotransfection andimmunoprecipitation assays. For example, interaction of STIM1 proteinscan be detected by cotransfection of cells (e.g., human 293T cells) withnucleic acid encoding human STIM1 and nucleic acid encoding a fusionprotein containing a detectable marker (e.g., the extracellular regionof the GCSF (granulocyte colony stimulating factor) receptor) proteinfused to the transmembrane and cytoplasmic (i.e., C-terminal) region ofhuman STIM1 and immunoprecipitation of the homo-multimer from cellularlysates with an anti-marker protein antibody followed by immunoblottingof the immune complexes with antibody that recognizes the C-terminalregion of STIM1. Similarly, interaction of STIM1 and STIM2 proteins canbe detected by cotransfection of cells (e.g., human 293T cells) withnucleic acid encoding STIM1 and STIM2 and immunoprecipitation of thehetero-multimer from cellular lysates with an antibody that recognizesSTIM1 or STIM2 and immunoblotting of the complexes with an antibody thatrecognizes both STIM1 and STIM2. Thus, an agent that modulates (e.g.,disrupts, inhibits or alters) interactions of STIM proteins can beidentified by assaying agents for an effect on the immune complex (e.g.,a decrease or elimination of the complex).

Antibodies that recognize STIM proteins (and/or portions thereof) aredescribed herein and/or known in the art and can be produced usingstandard techniques known to those of skill in the art. For example,antibodies generated against human STIM1 include a monoclonal antibodyprepared to amino acids 25-139 of human GOK (STIM1) (TransductionLaboratories, San Diego, Calif., U.S.A., catalog no. G72120), apolyclonal antibody prepared in sheep against an N-terminal peptide(amino acids 22-37) of human STIM1 (LSHSHSEKATGTSSG-C) with cysteineadded for conjugation, and a polyclonal antibody prepared in rabbitsagainst a C-terminal peptide (amino acids 657-685) of human STIM1(C-DNGSIGEETDSSPGRKKFPLKIFKKPLKK) with cysteine added for conjugation(see, e.g., Williams et al. (2001) Biochem. J. 357:673-685). Antibodiesgenerated against human STIM2 include peptide affinity-purifiedantibodies prepared by immunizing sheep with a 22-amino-acid peptidebased on the C-terminal region of human STIM2 (CHNGEKSKKPSKIKSLFKKKSK)(see, e.g., Williams et al. (2001) Biochem. J. 357:673-685). Antibodypools reactive with mammalian and invertebrate STIM proteins includePan-STIM antibodies prepared against multiple peptides simultaneously insheep. In one example, four peptides have been used to prepare aPan-STIM antibody pool: (1) HKLMDDDANGDVDVEESDEFLR-COOH (SEQ ID NO: 101;human STIM1), (2) HKQMDDDKDGGIEVEESDEFIR-COOH (SEQ ID NO: 102; humanSTIM2), (3) HRQLDDDDNGNIDLSESDDFLR-COOH (SEQ ID NO: 103; D. melanogasterSTIM and (4) HRDMDDDHSGSIDRNESFQFMK-COOH (SEQ ID NO: 104; C. elegansSTIM) (see, e.g., Williams et al. (2001) Biochem. J. 357:673-685).

(b) Level and Activity Assays

A number of in vitro and cell-based assays are known in the art and canbe modified as needed by one of skill in the art to identify agents thatalter the level and/or activity of proteins. The assay used can dependon the protein of interest and its activities. Thus, for example, if theprotein of interest is an ion transport protein, one method forassessing activity is the analysis of the electrophysiologicalproperties of the channel activity using procedures described herein andknown in the art. If the protein of interest has an enzymatic activity,then one method for assessing activity is the analysis of substrateinteraction and reaction. For example, if the protein of interest has aprotease activity, it typically can be assessed by analysis of substratecleavage. Potential test agents that modulate an activity of a proteininvolved in intracellular calcium modulation can be identified byevaluating the effect of such an agent on the particular activity of theprotein.

In a particular embodiment of these methods of screening for oridentifying an agent that modulates intracellular calcium, a proteinused in the method (or on which the method is based) is a STIM orSTIM-like protein (e.g., a STIM1, STIM2, DSTIM or CSTIM protein). Thetest agent can be one that modulates the level or activity of a STIM orSTIM-like protein or portion(s) thereof. Such agents can be any agentknown to modulate the level or activity of such proteins or can be oneidentified using methods described herein and/or known in the art.Activities of a mammalian STIM1 protein include binding of pre-B cellsand differentiated B lymphocytes (facilitated by divalent cations suchas Mn²⁺), augmentation of interleukin 7-dependent proliferation of pre-Bcells, and modulation of the morphology of 293T cells, i.e., a humanrenal carcinoma cell line transfected with large T antigen (see, e.g.,Oritani and Kincade (1996) J. Cell Biol. 134:771-782). Thus, an agentthat modulates an activity of mammalian STIM1 can be identified byassaying agents for an effect on any of these activities. Methods ofassaying for these activities include methods described by Oritani andKincade ((1996) J. Cell Biol. 134:771-782), such as a flowcytometry-based assay for binding of a human Ig-mouse STIM1 fusionprotein (soluble fusion protein purified on Protein A columns) to mousepre-B cells stained with an FITC-conjugated anti-Ig antibody, asemisolid agar colony-forming assay to enumerate IL-7 responsive Blymphocyte precursors (CFU-IL7), and a cell morphology-based assay using293T cells overexpressing mouse STIM1 through transfection with nucleicacid encoding full-length mouse STIM1. Thus, an agent that, for example,enhances or reduces (1) binding of STIM1 to pre-B cells, or (2) theaugmentation of B lymphocyte precursor proliferation by STIM1 or (3) theinduction of a rounded morphology and detachment of 293Tcellsoverexpressing STIM1 is an agent that modulates an activity of STIM.

Another activity of STIM1 is the suppression of tumor growth. Theexpression of human STIM1 (GOK cDNA) in rhabdomyoscarcoma and rhabdoidtumor cell lines RD and G401 induces growth arrest and degeneration(Sabbioni et al. (1997) Cancer Res. 57: 4493-4497). Thus, an agent thatmodulates an activity of STIM can be identified by assaying agents foran effect on this growth suppression activity. Methods for assaying thisactivity include methods described by Sabbioni et al. ((1997) CancerRes. 57: 4493-4497). Such methods include, for example, a cellmorphology- or phenotype-based assay using assessing rhabdomyosarcoma(e.g., A204, A673, Hs729, RD, SJCRH30, TE125.T and TE611 cells availablefrom the American Type Culture Collection (ATCC)) or rhabdoid tumorcells (e.g., G401 cells available from ATCC) transfected with nucleicacid encoding full-length human STIM1. Thus, an agent that, for example,enhances or reduces the STIM-dependent induction of thegrowth-suppressed phenotype (e.g., rounded and detached or enlarged anddegenerating cells) of such cells is one that modulates an activity ofSTIM.

A further activity of STIM proteins is modulation of the Notch signalingpathway. Cell-cell signaling mediated by activation of the Notchreceptor results in proteolytic cleavage of the intracellular domain ofNotch which translocates to the nucleus where it associates withDNA-binding proteins and activates gene expression which in turn affectsregulation of downstream target genes to influence cell differentiation,proliferation and apoptotic events. The modulation of Notch signaling bySTIM proteins can include a possible antagonistic effect based on thegeneration of a phenotype in transgenic Drosophila (overexpressingDSTIM) with similarity to Drosophila Delta and Notch mutants (see PCTApplication publication no. WO02/30976). Thus, an agent that, forexample, alters (e.g., enhances or reduces) the modulation of Notchsignaling by STIM proteins can be one that modulates an activity ofSTIM. One method for assessing whether an agent has an effect onSTIM-dependent modulation of Notch signaling involves contacting a cellthat exhibits altered Notch signaling due to overexpression of a STIMprotein with a test agent and determining whether the agent reverses, atleast partially, or enhances the STIM-dependent effect on or modulationof Notch signaling. Methods of monitoring Notch signaling are known inthe art (see, e.g., PCT Patent Application Publication No. WO02/12890,U.S. Pat. No. 6,436,650 and Karlstrom et al. (2002) J. Biol. Chem.277:6763-6766).

Potential test agents that modulate the levels and/or expression of aprotein involved in intracellular calcium modulation can be identifiedusing a number of techniques known in the art. For example, potentialagents that modulate the levels of expression of a protein can beidentified in cell-based assays in which the level of the protein(either an endogenous protein or a recombinant or reporter protein) isassessed upon exposure of the cell to a potential test agent. Thus, forexample, if the protein of interest is endogenously or recombinantlyexpressed in a cell using transcription regulatory sequences (e.g.,promoters, enhancers) from the gene that encodes the protein, it ispossible to identify test agents that modulate expression of the gene byevaluating protein levels of the cell. Alternatively, the transcriptionregulatory sequences of the gene can be operably linked to DNA encodinga readily measurable reporter protein and the levels of reporter proteinmeasured as an indication of the effect of a potential test agent onexpression levels of the protein of interest. Methods of measuringprotein levels are well known in the art, including, for example,quantitative electrophoretic analyses and immunoassays, e.g., ELISA andother assays (such as activity/property assays, e.g., reporter proteinactivity, fluorescence, bioluminescence). The levels of the mRNAtranscript of a protein of interest can also be determined upon exposureof a cell to a potential test agent to identify agents that modulateexpression of the protein. Methods of evaluating mRNA levels as anindication of levels of gene and/or protein expression are also wellknown in the art, including, for example, northern blotting and RT-PCR.

In a particular embodiment of these methods of screening for oridentifying an agent that modulates intracellular calcium, a test agentcan be one that modulates the level or expression of a STIM or STIM-likeprotein or portion(s) thereof. Such agents can be identified in a numberof ways. For example, the effect of a potential test agent on the levelof STIM mRNA or protein in a cell expressing a STIM protein (e.g.,endogenously or recombinantly) can be assessed to identify an agentassociated with changes in protein or mRNA levels relative to a cellthat has not been exposed to the potential test agent. Reagents (e.g.,STIM mRNA-specific nucleic acid probes and primers and STIM-specificantibodies) and techniques are described herein and/or known in the artfor detecting STIM mRNA and protein. Alternatively, a reportermolecule-based assay can be used to identify agents that modulateexpression of the gene and protein. A STIM gene promoter sequence can beoperably linked to a reporter molecule (e.g., nucleic acid encodingluciferase, a fluorescent protein, an enzyme that catalyzes a reactionthat yields a readily detectable product) and expressed in a host cellwhich is contacted with or exposed to potential test agents to monitoror assess the effect of agents on reporter molecule levels. In oneexample, a STIM gene promoter sequence can be cloned upstream of DNAencoding luciferase in the vector pGL3-Basic (Promega, Madison, Wis.)(see, e.g., vector pGL3-STIM1.B described by Sabbioni et al. (1999)Cytogenet. Cell Genet. 86:214-218). STIM gene promoter sequences includethe human STIM1 (GOK) gene promoter sequence (see, e.g., GenBankaccession no. AF139917 and Sabbioni et al. (1999) Cytogenet. Cell Genet.86:214-218).

ii. Monitoring or Assessing the Effects of Test Agent on IntracellularCalcium

In this embodiment of the methods of screening for or identifying anagent that modulates intracellular calcium, the effects of a test agent(which is one that binds to, interacts with and/or modulatesinteractions, activities, levels or any physical, structural or otherproperty of a protein involved in modulating intracellular) onintracellular calcium are monitored or assessed. A test agent isidentified as an agent that modulates intracellular calcium if it has aneffect on intracellular calcium. In particular embodiments, the effectof test agent on store-operated calcium entry, calcium levels in ormovement of calcium into, within or out of an intracellular organelle orcalcium store (e.g., endoplasmic reticulum) cytosolic calcium bufferingand/or resting cytosolic calcium levels is monitored or assessed.

Generally, in monitoring or assessing the effect of a test agent onintracellular calcium in any of the screening/identification methodsprovided herein, including this particular embodiment, some direct orindirect evaluation or measurement of cellular (including cytosolic andintracellular organelle or compartment) calcium and/or movement of ionsinto, within or out of a cell, organelle, or portions thereof (e.g., amembrane) is conducted. A variety of methods are described herein (seedetailed descriptions provided below and elsewhere herein) and/or knownin the art for evaluating calcium levels and ion movements or flux, andfor monitoring calcium entry-mediated events. The particular method usedand the conditions employed can depend on whether a particular aspect ofintracellular calcium is being monitored. For example, as describedherein, reagents and conditions are known, and can be used, forspecifically evaluating store-operated calcium entry, calcium levels ormovement of calcium into, out of or within an intracellular organelle orcalcium store, calcium buffering and resting cytosolic calcium levels.The effect of test agent on intracellular calcium can be monitored orassessed using, for example, a cell, an intracellular organelle orstorage compartment, a membrane (including, e.g., a detached membranepatch or a lipid bilayer) or a cell-free (e.g. outside-out membranevesicle) assay system. Generally, some aspect of intracellular calciumis monitored or assessed in the presence of test agent and compared to acontrol, including intracellular calcium in the absence of test agent.

In a particular embodiment of these methods, the effect of test agent onintracellular calcium is monitored or assessed using a cell. The cellcan be one that contains the particular protein involved inintracellular calcium modulation that the test agent binds to, interactswith, and/or modulates (e.g., the interactions, activities, levels orany physical, structural or other property of the protein).Alternatively, the cell can be one that does not contain the particularprotein involved in intracellular calcium modulation. If the test agentis a cellular protein, for example a cellular protein that binds to aprotein involved in modulating intracellular calcium, then the effect ofthe test agent on intracellular calcium can also be assessed by alteringthe expression or level of the cellular protein in a cell that expressesthe protein and monitoring or assessing the effect of the alteration onintracellular calcium. Methods for altering protein expression and/orlevels in a cell are described herein and/or known in the art.

In one embodiment of these methods, the cell used in the method is acell that exhibits store-operated calcium entry, and the effect of thetest agent on store-operated calcium entry is monitored or assessed.

d. Methods Based on Assessing the Interaction of a Test Agent with, orthe Effect of a Test Agent on, a Protein Involved in ModulatingIntracellular Calcium

In one embodiment of the methods for screening for or identifying anagent that modulates intracellular calcium provided herein, the testagent is one that modulates intracellular calcium. In particularembodiments, the test agent is one that modulates store-operated calciumentry, calcium levels in or movement of calcium into, within or out ofan intracellular organelle (or calcium store, e.g. endoplasmicreticulum), cytosolic calcium buffering and/or resting cytosolic calciumlevels. This embodiment of the methods includes a step of assessing theinteraction of the test agent with, or the effect of the test agent on,a protein involved in modulating intracellular calcium. A test agent isidentified as one involved in modulating intracellular calcium if itinteracts with and/or otherwise affects a protein involved in modulatingintracellular calcium. Thus, this embodiment of thescreening/identifying methods can be used to identify intracellularcalcium-modulating agents that modulate calcium through a process thatinvolves and that may be specific for an interaction or effect on aparticular protein or proteins. Test agents suitable for use in thisembodiment of the screening/identification methods can be agents alreadyknown to modulate intracellular calcium or can be identified as such ina number of ways using techniques described herein and/or known in theart for evaluating intracellular calcium. Agents that have been reportedto modulate intracellular calcium include, for example,stearoylethanolamide (SEA; see, e.g., Maccarrone et al. (2002) Biochem.J. 366:137-144) and reported store-operated calcium entry inhibitors(for example, statins in the δ-lactone form (e.g., lovastatin,mevastatin and simvastatin) and other compounds (see, e.g., PCT PatentApplication Publication WO02/096416)). The method can optionally includea step of identifying a test agent that modulates intracellular calciumprior to (or concurrently with) the step of assessing the interaction ofthe test agent with, or the effect of the test agent on, a proteininvolved in modulating intracellular calcium.

The protein involved in modulating intracellular calcium that is used inassessing a test agent with respect to whether the agent interacts withor affects the protein can be a protein as provided herein and describedabove (and elsewhere herein). In particular embodiments, the protein isone that is involved in, participates in, and/or provides forstore-operated calcium entry, movement of calcium into, out of or withinan intracellular organelle or calcium store, modulation of intracellularorganelle (or calcium store) calcium level, and/or cytosolic calciumbuffering. In particular embodiments, the protein is one of the proteins(or is substantially homologous to one of the proteins) listed in Table3. The protein can be a STIM or STIM-like protein, including a STIM1,STIM2, DSTIM or CSTIM protein. In one embodiment of the methods, theprotein is a STIM1 protein, for example, a mammalian, such as a human,STIM1 protein. In a particular embodiment, the protein is one that isinvolved in, participates in and/or provides for store-operated calciumentry.

As described above, a protein involved in modulating intracellularcalcium that is used in the methods for screening for or identifying anagent that modulates intracellular calcium (or a protein on which themethod is based) can be a full-length or complete protein or a portionof a complete protein. Portions of complete proteins include portionsthat are associated with, exhibit or that are sufficient for producing aparticular activity or function of the complete protein, and portionsthat are associated with a particular property or feature of thecomplete protein, such as a binding portion or a particular domain, orany random portion of the protein. Similarly, nucleic acids that encodeproteins that can be used in the methods (or on which a method is based)can encode a complete protein or portion(s) thereof.

In this embodiment of the methods that involves assessing a test agentfor interaction with, or effects on, a protein involved in modulatingintracellular calcium, the assessment can be conducted using a completeor full-length protein involved in modulating intracellular calcium orportion(s) thereof. Portions of proteins can be obtained using a varietyof methods (e.g., synthetically, through recombinant production methods)described herein and/or known in the art and can be random or selectedfor a particular feature. Methods for identifying particular features ofproteins are described herein and/or known in the art.

The test agent can be evaluated for any effect on a protein involved inmodulating intracellular calcium. For example, the test agent can beassessed for binding to a protein, interaction with a protein, ormodulation of interactions, activities, levels or any physical,structural or other property of a protein involved in modulatingintracellular calcium. Reagents and procedures for assessing proteinbinding, interactions, activities, levels and other protein propertiesare described herein (see, e.g., above sections entitled “Binding andinteraction assays” and “Level and activity assays”) and/or known in theart. For example, methods for assessing binding or interaction of a testagent with a protein involved in modulating intracellular calciuminclude NMR, mass spectroscopy, fluorescence spectroscopy, scintillationproximity assays, surface plasmon resonance assays and others. Examplesof methods for assessing modulation of interactions, activities, levelsor any physical, structural or other property of a protein involved inmodulating intracellular calcium include, but are not limited to, FRETassays to assess effects on protein interactions, NMR, X-raycrystallography and circular dichroism to assess effects on proteininteractions and on physical and structural properties of a protein, andactivity assays suitable for assessing a particular activity of aprotein.

In a particular embodiment of these methods of screening for oridentifying an agent that modulates intracellular calcium, a proteinused in the method (or on which the method is based) is a STIM orSTIM-like protein (e.g., a STIM1, STIM2, DSTIM or CSTIM) or portionthereof. Any portion of a STIM or STIM-like protein can be used in themethods, including the complete protein, the mature protein (e.g.,lacking a signal sequence) and any portion thereof. Particular domainsof STIM and STIM-like proteins are described herein and/or can beidentified using methods described herein and/or known in the art.Examples of nucleic acids encoding such proteins and portions thereofare also described herein or can be designed based on amino acidsequences such as those described herein. Methods of assessing bindingor interaction of an agent with a STIM protein and modulation of STIMprotein interactions or activities are described herein. For example, amethod for assessing the effect of a test agent on STIM proteinhomotypic or heterotypic interactions can include evaluating STIMprotein immune complex formation and/or levels in lysates of cells thatexpress (e.g., endogenously or recombinantly) STIM proteins (and/or STIMfusion proteins). Thus, for example, a test agent identified as one thatmodulates intracellular calcium in this embodiment of the methods can beone that disrupts, inhibits, enhances or otherwise alters STIM proteincomplexes as can be detected in immunoprecipitation assays. Methods forassessing the effect of a test agent on STIM protein activity caninclude, for example, evaluating the effect of a test agent on STIMprotein binding to pre-B cells and differentiated B lymphocytes, STIMaugmentation of IL-7-dependent proliferation of pre-B cells, STIMmodulation of cell morphology and STIM suppression of tumor growth.Thus, for example, a test agent identified as one that modulatesintracellular calcium in this embodiment of the methods can be one thatreduces, enhances or otherwise alters these activities of STIM proteins.Examples of particular reagents and procedures that can be used inassessing such activities are described herein. Methods of assessing theeffect of a test agent on STIM protein levels or expression can include,for example, evaluating the effect of a test agent on the level of STIMmRNA or protein in a cell expressing a STIM protein (e.g., endogenouslyor recombinantly) or on the level of reporter molecule or activity in acell expressing a STIM gene promoter sequence operably linked to anucleic acid encoding a reporter molecule. Thus, for example, a testagent identified as one that modulates intracellular calcium in thisembodiment of the methods can be one that reduces, enhances or otherwisealters expression of STIM protein or mRNA or of a reporter molecule.Examples of particular reagents and procedures that can be used inassessing protein and mRNA expression are described herein.

e. Methods Based on Testing the Effect of any Test Agent onIntracellular Calcium

In another embodiment of the methods for screening for or identifyingagents that modulate intracellular calcium provided herein, the effectof any test agent on intracellular calcium (and, in particularembodiments, the effect on store-operated calcium entry, calcium levelsin (and/or movement of calcium into, out of or within) intracellularorganelles or calcium stores (e.g., endoplasmic reticulum), cytosoliccalcium buffering and/or resting cytosolic calcium levels) is monitored,assessed or evaluated. In this embodiment of the methods, the test agentcan be any agent, and is not necessarily one that (or has beenidentified as one that) binds to, interacts with and/or modulates (e.g.,interactions, activities, levels or any physical, structural or otherproperty) a protein involved in modulating intracellular calcium.Generally, in this embodiment, the method can be performed by contactingany test agent with (1) one or more proteins involved in modulatingintracellular calcium (e.g. an ion transport protein, a component of anion transport protein complex, or a modulatory protein) and/or (2) acell, or portion thereof, e.g., a membrane, containing one or moreproteins involved in modulating intracellular calcium and/or nucleicacid (e.g., a gene or coding sequence such as cDNA or RNA), orportion(s) thereof, encoding such proteins. The one or more proteinsinvolved in modulating intracellular calcium that are used in thesemethods can be proteins as provided herein and described above (andelsewhere herein). In particular embodiments, the one or more proteinsis (are) one that is involved in, participates in, and/or provides forstore-operated calcium entry, movement of calcium into, out of or withinan intracellular organelle or calcium store, modulation of calciumlevels in an intracellular organelle or calcium store (e.g., endoplasmicreticulum) and/or calcium buffering. In particular embodiments, theprotein(s) is (or is substantially homologous to) one of the proteinslisted in Table 3. The protein can be, for example, a STIM or STIM-likeprotein (including a STIM1, STIM2, DSTIM and CSTIM protein). In oneembodiment, the protein is a STIM1 protein, for example, a mammalian,such as human or rodent, STIM1 protein. In a particular embodiment ofthe methods, the effect of test agent on store-operated calcium entry ismonitored or assessed.

As described above, a protein involved in modulating intracellularcalcium that is used in the methods for screening for or identifying anagent that modulates intracellular calcium (or a protein on which themethod is based) can be a full-length or complete protein or a portionof a complete protein. Portions of complete proteins include portionsthat are associated with, exhibit or that are sufficient for producing aparticular activity or function of the complete protein. The activity orfunction of the complete protein is one that is involved in modulatingintracellular calcium. Similarly, nucleic acids that encode proteinsthat can be used in the methods (or on which a method is based) canencode a complete protein or portion(s) thereof.

The monitoring, evaluation or assessment of intracellular calcium inthese embodiments of the methods can be conducted in a variety of wayswhich can be used for all embodiments of the screening/identificationmethods as described herein. The monitoring or assessing typicallyinvolves some direct or indirect evaluation or measurement of cellular(including cytosolic and intracellular organelle or compartment) calciumand/or movement of ions into, within or out of a cell, organelle, orportions thereof (e.g., a membrane). A variety of methods are describedherein (see detailed descriptions provided below and elsewhere herein)and/or known in the art for evaluating calcium levels and ion movementsor flux. The particular method used and the conditions employed candepend on whether a particular aspect of intracellular calcium is beingmonitored or assessed. For example, as described herein, reagents andconditions are known, and can be used, for specifically evaluatingstore-operated calcium entry, calcium levels or movement of calciuminto, out of or within an intracellular organelle or calcium store,calcium buffering and resting cytosolic calcium levels. The effect oftest agent on intracellular calcium can be monitored or assessed using,for example, a cell, an intracellular organelle or storage compartment,a membrane (including, e.g., a detached membrane patch or a lipidbilayer) or a cell-free (e.g., outside-out membrane vesicle) assaysystem. Generally, some aspect of intracellular calcium is monitored orassessed in the presence of test agent and compared to a control, e.g.,intracellular calcium in the absence of test agent.

Generally, a test agent is identified as an agent, or candidate agent,that modulates intracellular calcium if there is a detectable effect ofthe agent on intracellular calcium levels and/or ion movement or flux,such as a detectable difference in levels or flux in the presence of thetest agent. A test agent can also be identified as an agent, orcandidate agent, that modulates intracellular calcium if there is adetectable effect of the agent on a calcium-entry mediated event.Effects of a test agent on a calcium entry-mediated event can bedetected using assays known in the art, including assays such asprovided and described herein. In particular embodiments, a test agentis identified as one that produces at least a 50% difference in anyaspect or parameter of intracellular calcium (e.g., store-operatedcalcium entry, calcium buffering, calcium levels of or movement into,out of or within an intracellular organelle or calcium store) relativeto control (e.g., absence of compound, i.e., vehicle only). Inparticular embodiments, the effect or differences can be substantial orstatistically significant.

2. Methods of Identifying Molecules Involved in Modulating IntracellularCalcium

Proteins (and/or nucleic acids encoding proteins) involved in modulatingintracellular calcium, as described herein, further provide the basisfor additional methods of identifying molecules involved in modulatingintracellular calcium, as well as for methods of elucidating pathways,and elements thereof, of intracellular calcium modulation. Once aprotein has been identified as one involved in modulating intracellularcalcium, it can be used to identify molecules, in particular cellularcomponents, that interact with or bind to it and potentially function inthe modulation of intracellular calcium. Cellular components that can bemodulators of intracellular calcium include, but are not limited to,molecules such as proteins (e.g., enzymes, receptors, modulatoryproteins, ion transport proteins), nucleic acids, lipids and solublesecond messengers (e.g., IP₃, cADPR, cGMP and cAMP). Additionally, theidentification of molecules that interact with proteins involved inintracellular calcium modulation, facilitates the dissection andelucidation of pathways and mechanisms of cellular calcium regulationand signaling. The elucidation of such pathways provides additionaltargets that can be modulated in methods of modulating intracellularcalcium and for use in methods of identifying agents for modulatingintracellular calcium. Because the dissection of such pathways alsoelucidates components of the pathway and interactions between thecomponents, it further makes possible the refinement of methods ofmodulating intracellular calcium by the balanced targeting of modulationof one or more components and/or interactions of the pathway.

Particular proteins (and/or nucleic acids encoding proteins) involved inmodulating intracellular calcium (and, in particular, proteins that areinvolved in, participate in and/or provide for store-operated calciumentry, movement of calcium into, out of or within an intracellularcalcium store or organelle, modulation of calcium levels inintracellular calcium stores or organelles, and/or cytosolic calciumbuffering) are provided herein. These proteins can thus be used inmethods of identifying molecules, and, in particular, cellularcomponents, that interact with or bind to the proteins and that may beinvolved in modulating intracellular calcium.

Provided herein are methods of identifying a molecule or candidatemolecule involved in modulating intracellular calcium (and, inparticular, a molecule that is involved in, participates in and/orprovides for store-operated calcium entry, movement of calcium into, outof or within an intracellular calcium store or organelle, modulation ofcalcium levels in intracellular calcium stores or organelles, and/orcytosolic calcium buffering). The methods are useful in identifying newmolecules, e.g., proteins and/or nucleic acids encoding new proteins,that were previously unknown, and for identifying known molecules (e.g.,proteins) as being involved in intracellular calcium modulation. Themethods include a step of assessing the effect on intracellular calciumof a candidate or test molecule (e.g., a cellular component, such as aprotein) that interacts with a protein involved in modulatingintracellular calcium, or portion thereof. Assessing the effect onintracellular calcium can involve assessing the effect that modulationof the molecule and/or its interaction with the protein has onintracellular calcium. The methods can optionally include a step ofidentifying a candidate or test molecule, such as, for example, acellular component, that interacts with a protein involved inintracellular calcium modulation, or portion thereof.

Generally, in assessing the effect of a candidate or test molecule onintracellular calcium, some direct or indirect evaluation or measurementof cellular (including cytosolic and intracellular organelle or calciumstore) calcium and/or movement of ions into, within or out of a cell,calcium store, organelle, or portions thereof (e.g., a membrane) isconducted. A variety of methods are described herein (see detaileddescriptions provided above and elsewhere herein) and/or known in theart for evaluating calcium levels and ion movements or flux. Theparticular method used and the conditions employed can depend on whethera particular aspect of intracellular calcium is being monitored orassessed. For example, as described herein, reagents and conditions areknown, and can be used, for specifically evaluating store-operatedcalcium entry, calcium levels or movement of calcium into, out of orwithin an intracellular organelle or calcium store, calcium bufferingand resting cytosolic calcium levels. The effect of a candidate or testmolecule on intracellular calcium can be monitored using, for example, acell, an intracellular organelle, calcium store or storage compartment,a membrane (including, e.g., a detached membrane patch or a lipidbilayer) or a cell-free (e.g., outside-out membrane vesicle) assaysystem.

Generally, in determining whether a test or candidate molecule has aneffect on intracellular calcium, some aspect of intracellular calciumcan be compared under differing conditions of the test molecule. Thus,for example, an interaction of, an activity of and/or the level or sizeof the test molecule can be altered or modulated and intracellularcalcium can be compared under the different conditions (i.e., unaltered(control) vs. altered interaction, activity, level or size of the testmolecule). In a particular embodiment of these methods, the effect of atest or candidate molecule on intracellular calcium is assessed using acell, or portion thereof. The cell, or portion thereof, can be one thatcontains the test molecule, or portion thereof, and may or may notcontain the particular protein involved in intracellular calciummodulation with which the test molecule binds to or interacts. Thus, forexample, one embodiment of the methods includes comparing intracellularcalcium of a control cell (or portion thereof) containing the testmolecule (or portion thereof) and intracellular calcium of a test cell(or portion thereof) that differs from the control cell by an alterationof an interaction, activity, size and/or level of the test moleculerelative to the control cell. In one embodiment, the interaction betweena test molecule (or portion thereof) and the protein with which itinteracts (or portion thereof) is altered in the test cell (or portionthereof) relative to a control cell (or portion thereof).

a. Proteins Involved in Intracellular Calcium Modulation

Methods of identifying a molecule involved in modulating intracellularcalcium provided herein involve assessing the effect of a candidatemolecule that interacts with a protein involved in modulatingintracellular calcium (or portion thereof) on intracellular calcium. Theprotein can be a protein involved in modulating intracellular calcium asprovided herein and described above (and elsewhere herein). Thus, forexample, the protein can be one that is homologous to an amino acidsequence of the protein encoded by the coding sequence of Drosophilagene CG9126 and/or to a mammalian stromal interacting molecule (STIM)protein, e.g., human or rodent (such as rat) STIM1. In particularembodiments, the protein is one that is involved in, participates inand/or provides for store-operated calcium entry, movement of calciuminto, out of or within an intracellular calcium store or organelle,modulation of calcium levels in intracellular calcium stores ororganelles, and/or cytosolic calcium buffering. In particularembodiments, the protein is one of the proteins (or is substantiallyhomologous to one of the proteins) listed in Table 3. The protein canbe, for example, a STIM or STIM4-like protein, including a STIM1, STIM2,DSTIM or CSTIM protein. In one embodiment of the methods, the protein isa STIM1 protein, for example, a mammalian STIM1 protein.

The protein involved in modulating intracellular calcium with which acandidate or test molecule interacts or binds can be a complete protein(e.g., the entire amino acid sequence encoded by a gene or mRNAtranscript which can include a signal sequence), a mature, processedprotein (e.g., lacking signal sequence) or any fragment or portion of acomplete or mature protein that is made up of less than all of the aminoacid sequence of a complete or mature protein can be used. Portions ofcomplete proteins include portions that are associated with, exhibit orthat are sufficient for producing a particular activity or function ofthe complete protein, and portions that are associated with a particularproperty or feature (e.g., structural feature) of the complete protein,such as a binding portion or a particular domain. Domains thus include,for example, beta sheets, alpha helices, loops, folds, hydrophobicdomains, amphipathic domains, extracellular, cytoplasmic, intralumenaland transmembrane domains, an epitope, a ligand- or effector-bindingsite, a modification site (e.g., a site of phosphorylation, acylation,or glycosylation), and an enzyme active site. There are numerous methodsknown in the art for identifying domains within a protein. For example,transmembrane domains can be identified through analysis of hydropathyplots of the hydrophobicity of adjacent amino acid residues (commonlyused to assess proteins for transmembrane domains; see, e.g., Kyte andDoolittle (1982) J. Mol. Biol. 157:105-134; Argos et al. (1982) Eur. J.Biochem. 128:565-575 and Engelman et al. (1984) Ann. Rev. Biophys.Biophys. Chem. 15:321-353) and through the use of programs such asTMpred (see, e.g., www.ch.embnet.org/software/TMpred_form.html which canbe accessed via the ExPASy Molecular Biology Server (www.expasy.ch)).The TMpred program predicts membrane-spanning regions and theirorientation through use of an algorithm generated by statisticalanalysis of naturally occurring transmembrane proteins (i.e., proteinscontained in the TMbase database; see Hofman and Stoffel (1993) Biol.Chem. Hoppe-Seyler 374:166). Domains can also be identified by homologyto proteins known to contain particular structural or functionaldomains, for example, by using the search and alignment resources ofconserved domain databases, such as the Pfam database (pfam.wustl.edu/;Bateman et al. (2002) Nuc. Acids Res. 30:276-280), the Simple ModuleArchitecture Research Tool (SMART; accessible throughsmart.embl-heidelberg.de; see also Letunic et al. (2002) Nuc. Acids Res.30:242-244) and the Clusters of Orthologous Groups (COG) database (aphylogenetic classification of proteins from complete genomes accessiblethrough www.ncbi.nlm.nih.gov/cog; see also Tatusov et al. (2001) Nuc.Acids Res. 29:22-28) which groups proteins into clusters (or COGs) ofvery similar proteins with an ancient conserved domain found in at leastthree species. These databases of conserved domains assign identifyingnumbers (which begin with database identifiers such as “Pfam,” “smart”and “COG”) to particular domains that have been characterized inproteins.

Particular portions of a STIM protein with which a candidate or testmolecule can interact or bind include, but are not limited to, thefollowing domains: N-terminal (putative extracellular), transmembrane,C-terminal (putative cytoplasmic), signal peptide, N-terminal closelyspaced cysteine residues, EF hand, sterile α-motif (SAM) and coiled-coildomains. Particular portions of STIM1 and STIM2 proteins that maydistinguish them from DSTIM proteins include a consensus sequence (YYNI)for phosphorylation-dependent binding of Src homology type 2 (SH2)domains, proline/serine-rich (STIM1) or proline/histidine-rich (STIM2)domains, potential SH3 domain binding motifs (PXXP) and a lysine-richdomain. In contrast, DSTIM contains a myosin tail domain not present inSTIM1 or STIM2 as well as an N-terminal domain that has no equivalent inSTIM1 or STIM2. Particular portions of a STIM1 protein that maydistinguish it from a DSTIM or STIM2 protein include an N-linkedglycosylation site within the SAM domain, a dibasic proteolytic cleavagesite, ATP synthase B/B□ domain (pfam00430), ezrin/radixin/moesin (ERM;pfam00769) domain and a diacylglycerol kinase accessory (DAGKa;smart00045) domain. A particular portion of a STIM2 protein that maydistinguish it from a DSTIM or STIM1 protein is an SMC domain (COG1196).Table 4 provides examples of STIM protein domains with reference toparticular Drosophila DSTIM, human and rat STIM1, and human STIM2proteins. TABLE 4 STIM Protein Domains DSTIM (SEQ ID NOS: 2, HUMAN HUMAN76, 78, STIM1 (SEQ Reference STIM2 (SEQ 80, 81) ID NOS: 4, STIM1 (SEQ IDNOS: 6, Approx- 83, 84) ID NO: 52) 87, 88) imate Approximate ApproximateApproximate Amino Amino Acid Amino Acid Amino Acid Acid DOMAIN PositionsPositions Positions Positions N-Terminal  1-212  1-213  1-217  1-294N-Terminal  23-212  23-213  15-217  24-294 (without signal peptide)Transmembrane 213-234 214-234 218-235 295-312 C-Terminal 235-685 235-685236-746 313-570 Signal Peptide  1-22  1-22  1-14  1-23 N-Terminal 27-125 Subdomain Closely Spaced 49-56 49-56 53-60 126-133 CysteineResidues EF Hand 76-87 76-87 80-91 155-166 Sterile α-motif 129-196132-200 (SAM) 136-204 213-281 Dibasic 207-209 207-209 ProteolyticCleavage Site Coiled-Coil 238-343 and 238-343 and 242-344 and 310-407362-390 362-390 358-394 and 420-462 Coiled-Coil and 238-424 238-424 ERMConsensus 361-364 361-364 365-368 Sequence for SH2 Domain BindingProline/Serine-or 600-629 600-629 533-559 Proline/Histidine- Rich SH3Domain- 573-629 573-629 521-559 Binding Region Lysine-Rich 672-685672-685 730-746 Proline/Serine- 591-685 591-685 and Lysine-Rich MyosinTail 324-491 Domain ATP synthase 249-337 249-337 B/B□ domain (pfam00430)ERM 253-424 253-424 (pfam00769) DAGKa 422-484 422-484 (smart00045) SMCdomain 238-340 (COG1196)

A portion or fragment of a protein can be obtained using a number ofprocesses known in the art. For example, a portion of a protein can beobtained by synthetic methods, by degradation of a complete protein andby recombinant expression of a nucleic acid encoding the amino acidsequence of the portion.

A candidate or test molecule, such as a cellular component, thatinteracts with or binds to a protein involved in modulatingintracellular calcium, or portion thereof, can be one that is known tointeract with or bind to such a protein (or portion thereof) or can beone that is identified (but not previously known) as one that interactswith or binds to such a protein (or portion thereof).

b. Identifying Candidate or Test Molecules

Candidate or test molecules (such as cellular components, e.g., aprotein) suitable for use in the methods for identifying a moleculeinvolved in modulating intracellular calcium can be identified in anumber of ways using techniques described herein and/or known in the artfor detecting an interaction or binding between a protein and amolecule. The interaction can be any direct or indirect physical,biochemical, chemical or other interaction between a molecule and aprotein (or portion thereof) involved in intracellular calciummodulation, such as those described herein. Interactions between amolecule and a protein (or portion thereof) involved in modulatingintracellular calcium include, for example, covalent, non-covalent,ionic, electrostatic, hydrophobic, hydrogen bonding, disulfide bondingand other interactions.

Several methods for identifying a molecule that interacts with a proteinare described herein with respect to the methods of screening for oridentifying agents that modulate intracellular calcium. For example,binding assays can be used to identify an interaction between a moleculeand a protein involved in intracellular calcium modulation, or portionthereof. A number of in vitro and cell-based binding assays are known inthe art and can be modified as needed by one of skill in the art toidentify molecules that bind to or interact with a protein (or portionthereof) involved in modulating intracellular calcium. In one suchassay, the protein (or portion thereof) involved in intracellularcalcium modulation is contacted with cellular components (within thecontext of a cell or in isolation), cell medium, a cell and/or a cellextract and assayed for binding of a molecule to the protein (or portionthereof) involved in modulating intracellular calcium. Binding can beevaluated and detected using any of several methods known in the art fordetecting binding of molecules to proteins, including, but not limitedto, affinity chromatography (including Biacore and Ciphergentechnologies), immunoprecipitation, ELISA assays, far-western blottingand other methods. Immunoassays to detect binding of molecules to aprotein involved in intracellular calcium modulation can utilizeantibodies (e.g., monoclonal and polyclonal) prepared against theprotein or portions thereof. Methods of generating and testingantibodies against proteins are well known in the art. Interactionsbetween a protein (or portion thereof) involved in intracellular calciummodulation and a candidate molecule involved in modulating intracellularcalcium can also be identified using assays such as co-purificationassays (e.g., GST pull-down assays, co-immunoprecipitation assay,chromatographic assays), phage display, ribozyme display, and proteinarrays. Detection of in vitro binding or interaction between a candidatemolecule and a protein involved in modulating intracellular calcium caninvolve a variety of approaches, such as, for example, nuclear magneticresonance (NMR), mass spectroscopy, fluorescence spectroscopy,scintillation proximity assays (SPQ), surface plasmon resonance assays(available commercially from BIACORE; www.biacore.se/proteomics/), andothers. Cell-based binding assays include, for example, yeast two-hybridassays (see, e.g., U.S. Pat. Nos. 5,283,173, 5,468,614 and 5,667,973 andPCT Application Publication Nos. WO01/25420 and WO02/079493); bacterialtwo-hybrid assays (Juong (2001) J. Cell Biochem. Suppl. 37:53-57), andothers. Such assays are particularly suitable for identifyingpolypeptides that interact with a protein involved in modulatingintracellular calcium. Another cell-based assay for identification ofmolecules that interact with a protein, such as a protein involved inmodulating intracellular calcium, is the tandem affinity purification(TAP) method (see, e.g., Rigaut et al. (1999) Nature Biotech.17:1030-1032; Puig et al. (2001) Methods 24:218-229).

Methods are also known in the art for identifying interaction of aprotein (or portion thereof) with a lipid. For example, centrifugationand FRET (fluorescence resonance energy transfer) assays can be used toassess protein binding to phospholipid vesicles (see, e.g., Ou-Yang etal. (Jan. 21, 2003) J. Biol. Chem. Manuscript M212606200; Bazzi andNelsestuen (1991) Biochemistry 29:7624-7630; Rietveld et al. (1986) J.Biol. Chem. 261:3846-3856). Immunoassays (such as ELISA assays) can alsobe used to assess binding of a protein to phospholipid (see, e.g., Ghoshet al. (1996) J. Biol. Chem. 271:8472-8480) or other cellularcomponents.

A molecule that has bound to or otherwise interacted with the protein(or portion thereof) involved in intracellular calcium modulation can becharacterized and identified using methods that are also well known inthe art, including, for example, HPLC, FPLC, amino acid sequencing ifthe molecule is a protein, and cloning of nucleic acid encoding amolecule that is a protein.

c. Assessment of the Effects of a Candidate or Test Molecule onIntracellular Calcium

A molecule that interacts or binds with a protein (or portion thereof)involved in modulating intracellular calcium is evaluated to assess itseffects on intracellular calcium. A candidate or test molecule (e.g., acellular component such as a protein) is identified as one thatmodulates intracellular calcium if it has an effect on intracellularcalcium. In particular embodiments, the effect of a candidate or testmolecule on store-operated calcium entry, calcium level in or movementof calcium into, out of or within an intracellular calcium store ororganelle, cytosolic calcium buffering and/or basal cytosolic calciumlevels is monitored or assessed.

There are a number of ways in which the effect of a candidate or testmolecule on intracellular calcium can be assessed. For example, a cell,or portion thereof (e.g., a membrane, intracellular organelle orintracellular calcium store) comprising the interacting molecule and theprotein involved in modulating intracellular calcium can be used forsuch evaluations. The interaction between the molecule and the proteininvolved in intracellular calcium modulation can be altered or disruptedor inhibited in the cell, or portion thereof, and then intracellularcalcium can be monitored to determine if intracellular calcium iseffected by the alteration in the interaction. Intracellular calcium canbe assessed using any methods known in the art and/or described herein.

In another process for assessing the effect of a candidate or testmolecule on intracellular calcium, the amount and/or activity of themolecule in a cell, or portion thereof, can be altered, for example,increased, decreased or the molecule can be eliminated in the cell, orportion thereof, and the effects of such an alteration or elimination onintracellular calcium can be assessed. For example, the synthesis and/ordegradation of the molecule can be altered (e.g., increased, reduced orinhibited) in the cell or portion thereof. One method for altering theamount of the molecule in the cell, if the molecule is a protein ornucleic acid produced by the cell, is to alter the expression of theprotein or nucleic acid in the cell. This can be accomplished using avariety of methods, including methods described herein and/or known inthe art, such as for example, RNA interference, antisense RNA methods,gene knock-out procedures and gene insertion/over-expression processes.Many such methods can require knowledge of at least a portion of thenucleotide sequence of the molecule (if the molecule is a nucleic acid)or of the gene or transcript or coding sequence encoding the molecule(e.g., if the molecule is a protein). If the nucleic acid or protein isone that is found in a cell of an organism for which much of the genomesequence is available, then at least a portion of the nucleotidesequence may be publicly available for use in altering the expression ofthe gene. If the nucleotide sequence of a gene (or portion of a gene)encoding a candidate or test molecule is not known, then cloning methodswell known in the art can be used to obtain at least a portion of thenucleotide sequence. It is also possible to assess the effect onintracellular calcium of a candidate or test molecule of one species byaltering expression of a homologous gene (e.g., an ortholog) of adifferent organism (e.g., an organism for which genomic sequence isreadily available) and assessing the effects of the alteration onintracellular calcium of a cell of the different organism.

i. Reduction, Alteration or Elimination of the Expression of a Gene in aCell

One process for assessing the effect of a candidate or test molecule onintracellular calcium involves reduction, alteration or elimination ofthe expression of a gene encoding the candidate molecule in a cell andthe assessment of intracellular calcium (e.g., intracellular/cytosoliccalcium levels and/or calcium movement into, within or out of the cell)to determine the effects of reduction, alteration or elimination of geneexpression on intracellular calcium (and, in particular, onstore-operated calcium entry, calcium levels in or movement of calciuminto, out of or within an intracellular calcium store or organelle,and/or cytosolic calcium buffering). A molecule (such as a protein) thatis involved in modulating intracellular calcium (and nucleic acidencoding such a protein) can be identified by a cell in which reduction,alteration or elimination of the expression of a gene encoding themolecule is accompanied by an alteration in intracellular calcium (e.g.,intracellular calcium levels and/or calcium ion movement into, out of orwithin a cell). Following and/or simultaneously with the alteration ofgene expression in a cell, the cell (or portion thereof) is analyzed toevaluate the effect, if any, on intracellular calcium.

These methods for assessing the effect of a candidate or test molecule,such as a protein (and/or nucleic acid encoding a protein), onintracellular calcium are function-based and specific; that is, theyprovide a direct, one-to-one correlation between a molecule that can beexpressed in its native cellular environment and cell calcium. Thecorrelation is determined by the association of altered expression of agene with alterations in intracellular calcium. In particularembodiments, a molecule involved in modulating intracellular calcium isidentified by a gene that, when expressed in an altered fashion(including a reduction or elimination of expression), results in alteredcytosolic calcium buffering, altered basal cytosolic calcium levels,altered calcium levels in an intracellular calcium store and/or altered,reduced or eliminated store-operated calcium entry ion or movement ofcalcium into, out of or within an intracellular calcium store. Themolecule, such as a protein, encoded by the gene is thereby identifiedas a molecule that is involved in, participates in and/or provides formodulation of calcium levels in or movement of calcium into, out of orwithin an intracellular calcium store or organelle, cytosolic calciumbuffering and/or store-operated calcium entry.

Techniques for altering gene expression in a cell are known in the artand described herein. Any such procedures may be used in the methods ofidentifying a molecule involved in modulating intracellular calciumprovided herein. With respect to these methods, the alteration in geneexpression need only be such that any associated alteration inintracellular calcium (e.g., intracellular calcium levels and/or calciummovement into, out of or within a cell) is detectable. An alteration ingene expression may be complete or nearly complete elimination ofexpression of a gene, a reduction in the expression of a gene, anincrease in the expression of a gene (overexpression), or an alterationin the protein encoded by the gene (such as a truncation or otheralteration that effectively renders the protein nonfunctional orprovides for aberrant functioning of the protein), e.g., relative to theexpression of the gene in a cell that has not been altered in itsexpression of the gene. Methods for assessing the type and extent of analteration in gene expression are known in the art. For example, thelevel and characteristics (e.g., size) of a transcript (e.g., mRNA)generated from a gene can be evaluated by Northern blot analysisemploying nucleic acid probes that specifically hybridize to thetranscript or by reverse transcriptase PCR (RT-PCR) nucleic acidamplification. The level and characteristics (e.g., size andimmunoreactivity) of a protein encoded by a gene can be evaluated bywestern blot or other immunoassays employing antibodies thatspecifically bind the protein.

For example, a complete or nearly complete elimination of expression ofa gene encoding a molecule that is principally involved in providing forstore-operated calcium entry in the cell may identify the molecule assuch by a complete or nearly complete elimination of store-operatedcalcium entry in a cell. If the molecule substantially participates inbut is not principally involved in providing for store-operated calciumentry, then elimination of the expression of the gene encoding themolecule may result in an alteration and/or reduction, but perhaps notelimination, of store-operated calcium entry in the cell. For example,if the molecule is an ion transport protein that is involved instore-operated calcium entry by being a component, such as a subunit, ofa multi-subunit complex (e.g., heteromeric complex) providing forstore-operated calcium entry, then complete or near complete eliminationof expression of the gene encoding the molecule may result in alteredstore-operated ion current properties, e.g., ion selectivity and/orconductance, reduced, or even increased store-operated entry into thecell. In another example, if expression of a gene is reduced, but noteliminated, and the gene encodes a molecule (e.g., a protein) that isprincipally involved in providing for store-operated calcium influx inthe cell, then the molecule may be identified as such by a partialreduction of store-operated calcium entry in the cell. If the geneencodes a molecule that substantially participates in but is notprincipally involved in providing for store-operated calcium entry, thenpartial elimination of expression of the gene may identify the moleculeas such by an alteration and/or reduction of store-operated calciumentry in the cell.

If the molecule is involved in modulation of intracellular calcium storecalcium levels or maintenance of resting cytosolic calcium levels, thenit may be identified as such, for example, by an alteration in calciumlevels (intracellular calcium store or resting cytosolic calcium levels)in a cell in which expression of the gene encoding the molecule has beenaltered or eliminated. An alteration of these calcium levels can be, forexample, a reduction, depletion, elimination of, or increase in calciumlevels, e.g., relative to the levels in a control cell (e.g., one thathas not been altered with respect to expression of the gene encoding themolecule).

If the molecule is involved in modulation of cytosolic calciumbuffering, then it may be identified as such, for example, by analteration in cytosolic calcium buffering in a cell in which expressionof the gene encoding the molecule has been altered or eliminated. Analteration in cytosolic calcium buffering can be, for example, acomplete or nearly complete elimination of the activity, a reduction ofthe activity, an alteration in properties or characteristics of theactivity (e.g., rates, kinetics or timing) or an increase in theactivity, e.g., relative to the activity in a control cell (e.g., a cellthat has not been altered with respect to expression of the geneencoding the molecule). Thus, for example, an alteration in calciumbuffering can be a reduction or increase in the rate at which cytosoliccalcium levels return to basal levels after activation of calcium influxinto the cytoplasm. The alteration can be an overall time course ofcytosolic calcium level adjustment that differs from that in a controlcell. In another example, an alteration in calcium buffering can be adelay in onset of the adjustment of cytosolic calcium levels to returnto basal levels after activation of calcium influx into the cytoplasm.In another example, an alteration in calcium buffering can be anadjustment in cytosolic calcium levels after activation of calciuminflux that does not result in a return of calcium levels to a basallevel, but instead to a level that is higher or lower than the basallevel. In yet a further example, an alteration in calcium buffering canbe a complete or near complete absence of an adjustment in cytosoliccalcium levels following activation of calcium influx into thecytoplasm.

Although any cell may be used in the methods, cells that areparticularly suitable are those that exhibit a number of calciumtransport processes and/or those in which calcium levels and/or movementmay readily be assessed. Choice of cell for use in the methods may alsodepend on whether a particular aspect of intracellular calcium (e.g.,store-operated calcium entry, calcium levels in or movement of calciuminto, out of or within an intracellular calcium store, and/or cytosoliccalcium buffering) is being evaluated to assess the effect of alteringgene expression. In instances in which a particular aspect ofintracellular calcium is assessed, a cell used in the method can be onewhich is particularly amenable to analysis of that aspect ofintracellular calcium or one that exhibits the particular aspect ofintracellular calcium modulation. For example, selection of a cell thatexhibits store-operated calcium entry is described herein and can beaccomplished using procedures known in the art.

Another feature of a cell that is particularly suitable for use in themethods that utilize alteration of gene expression for assessing theeffect of a candidate molecule on intracellular calcium is amenabilityto gene expression alteration. A number of techniques for altering geneexpression in cells are known in the art and described herein. Therelative ease with which these techniques may be applied to a cell toeffect reduction, alteration, increase or elimination of expression of agene in the cell is a consideration in selection of cells for use in themethods provided herein. Amenability to gene expression alteration andanalysis of ion flux particularly may be considerations.

(a) Antisense RNA Methods

Introduction into a cell of RNA complementary to an RNA transcriptencoded by a gene within the cell can be used to alter expression of thegene. Such an approach may be referred to as an “antisense” RNA methodwhen a single-stranded RNA molecule is introduced into a cell (see,e.g., Izant and Weintraub (1984) Cell 36:1007-1015). RNA can besynthesized from, for example, phagemid clones containing DNAcorresponding to a gene to be targeted for alteration of expression,using T3 and T7 polymerase. DNA templates may be removed by DNasetreatments. Antisense RNA is then introduced into a cell, and, after anappropriate period, intracellular calcium (e.g., intracellular calciumlevels and/or calcium movement into, out of or within the cell) isevaluated and compared to intracellular calcium prior to introduction ofantisense RNA or to intracellular calcium in a substantially similarcell that has not received antisense RNA. Antisense RNA may also beexpressed in a cell by transfecting the cell with a plasmid containingnucleic acid coding for antisense RNA.

(b) RNA Interference (RNAi) Methods

RNA interference (RNAi) is a method of gene silencing which involves theintroduction of double-stranded RNA (dsRNA) into cells. The basicpremise of RNAi is the ability of double-stranded RNA (dsRNA) tospecifically block expression of its homologous gene when present incells. Thus, in performing RNAi, a dsRNA construct containing anucleotide sequence with homology to or identical to a portion of thetarget gene to be silenced can be introduced into a cell (includingintroduction from outside of the cell and introduction by generating thedsRNA within a cell) containing the target gene. Generally, in the RNAireaction, both strands (sense and antisense) of the dsRNA are processedto small RNA fragments or segments of from about 21-23 nucleotides (nt)in length. Processing of the dsRNA to the small RNA fragments does notrequire the targeted mRNA, which demonstrates that the small RNA speciesis generated by processing of the dsRNA and not as a product ofdsRNA-targeted mRNA degradation. The mRNA is cleaved only within theregion of identity with the dsRNA. Cleavage occurs at sites 21-23nucleotides apart, the same interval observed for the dsRNA itself,suggesting that the 21-23 nucleotide fragments from the dsRNA areguiding mRNA cleavage. The RNAi phenomenon is mediated by a set ofenzyme activities that are evolutionarily conserved in eukaryotesranging from plants to mammals. After partial purification, amulti-component nuclease (RISC nuclease) co-fractionates with the dsRNAfragments which may confer specificity to the nuclease through homologyto the substrate mRNAs. It is believed that the dsRNA fragments instructthe RISC nuclease to destroy specific mRNAs corresponding to the dsRNAsequences. An additional enzyme, Dicer, has been identified that canproduce the guide RNAs. Dicer is a member of the RNAse III family ofnucleases that specifically cleave dsRNA and is evolutionarily conservedin worms, flies, plants, fungi and mammals. The enzyme has a distinctivestructure which includes a helicase domain and dual RNAse III motifs.Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTEfamily, which have been genetically linked to RNAi in lower eukaryotes.Activation, or overexpression, of Dicer and/or Argonaute is, thus,useful for facilitating RNAi in cells, such as cultured eukaryoticcells, or mammalian cells in culture or in whole organisms.

Mammalian cells exhibit an interferon-mediated antiviral response tolong dsRNA that results in diminished protein synthesis. This responsemakes it difficult to utilize long dsRNA in RNA interference ofmammalian cells. Thus, for RNAi of mammalian cells, short interferingdsRNAs of about 21 nucleotides can be used which do not activate theantiviral response (see, e.g., Elbashir et al. (2001) Nature411:494-498). Additionally, cells can be treated with an agent(s) thatinhibits the double-stranded RNA-dependent protein known as PKR (proteinkinase RNA-activated). Part of the interferon response is the activationof the PKR response. PKR phosphorylates and inactivates eIF2α.Inactivation of eIF2α results in inhibition of protein synthesis andultimately apoptosis. This sequence-independent PKR response can beovercome in favor of the sequence-specific RNAi response withoutaltering the activity of PKR; however, in certain instances, it may bedesirable to treat the cells with agents which inhibit expression ofPKR, cause its destruction, and/or inhibit the kinase activity of PKR.Likewise, overexpression of an agent which ectopically activates eIF2αcan be used.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. ThedsRNA construct may include modifications to either the phosphate-sugarbackbone or the nucleoside. The backbone may be modified for stabilityor for other reasons. The phosphodiester linkages may be modified toinclude at least one of a nitrogen or sulfur heteroatom. RNA duplexformation may be initiated either inside or outside the cell. The RNAmay be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

Double-stranded RNA constructs containing a nucleotide sequenceidentical to a portion of the target gene are generally most effectivein the inhibition of target gene expression. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence have also been found to be effective for inhibition. Sequenceidentity may be optimized by sequence comparison and alignmentalgorithms known in the art (see e.g., Gribskov and Devereux, SequenceAnalysis Primer, Stickton Press, 1991) and calculating the percentdifference between the nucleotide sequences by, for example, theSmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group). Greater than 90% sequence identity, or even 100%sequence identity, generally provides for the greatest inhibition;however, it is not required for inhibition. The RNAi method is able totolerate sequence variations that might be expected due to geneticmutation, strain polymorphism, or evolutionary divergence.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript. The length of the identical nucleotidesequences may be, for example, at least 25, 50, 100, 200, 300, or 400bases.

The dsRNA construct may be synthesized either in vivo or in vitro.Endogenous RNA polymerase of the cell may mediate transcription in vivo,or cloned RNA polymerase can be used for transcription in vivo or invitro. For transcription from a transgene in vivo or an expressionconstruct, a regulatory region (e.g., promoter, enhancer, silencer,splice donor and acceptor, polyadenylation) may be used to transcribethe dsRNA strand (or strands). Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. The RNA strands may or may not be polyadenylated; the RNA strandsmay or may not be capable of being translated into a polypeptide by acell's translational apparatus. The dsRNA construct may be chemically orenzymatically synthesized by manual or automated reactions. The dsRNAconstruct may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art (see, e.g., WO97/32016;U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and5,804,693). If synthesized chemically or by in vitro enzymaticsynthesis, the RNA may be purified prior to introduction into the cell.For example, RNA can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography or acombination thereof. Alternatively, the dsRNA construct may be used withno or a minimum of purification to avoid losses due to sampleprocessing. The dsRNA construct may be dried for storage or dissolved inan aqueous solution. The solution may contain buffers or salts topromote annealing, and/or stabilization of the duplex strands.

RNAi can be used to alter gene expression in a cell derived from orcontained in any organism. The organism may be a plant, animal,protozoan, bacterium, virus, or fungus. For example, such organismsinclude, but are not limited to Drosophila, trypanosomes, lanaria,hydra, zebrafish, Caenorhabditis elegans, mice, rats, humans and othermammals. The cell may be from, for example, the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, a stem cell or a differentiatedcell. The cell may be any individual cell of the early embryo, and maybe a blastocyte, or, alternatively, it may be an oocyte (see, e.g., Fireet al. (1998) Nature 391:806; Clemens et al. (2000) Proc. Natl. Acad.Sci. U.S.A. 97:6499-6503; PCT International Application Publication Nos.WO01/36646, WO99/32619, WO01/68836, WO01/29058 and WO01/75164).

The dsRNA may be directly injected into the cell or may be introduced bybathing the cell in a solution containing RNA. Other methods forintroducing dsRNA into a cell include bombardment by particles coveredby the RNA, for example gene gun technology in which the dsRNA isimmobilized on gold particles and fired directly at the site, andelectroporation of cell membranes in the presence of the RNA. Preciseconditions for electroporation depend on the device used to produce theelectro-shock and the dimensions of the chamber used to hold the cells.This method permits RNAi on a large scale. Any known gene therapytechnique can also be used to administer the RNA. A viral constructpackaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. In a particular example, alentivirus-based vector can be used to introduce siRNAs into primarycells (e.g., T lymphocytes) (see, e.g., Qin et al. (2003) Proc. Natl.Acad. Sci. U.S.A. 100:183-188). Other methods known in the art forintroducing nucleic acids into cells may be used, such as lipid-mediatedcarrier transport, chemical-mediated transport, such as calciumphosphate, and the like. Thus, the RNA may be introduced along withcomponents that perform one or more of the following activities: enhanceRNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or otherwise increase inhibition of thetarget gene. A transgenic animal that expresses RNA from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate animal.

RNAi may also be performed on an organismal level. Mammalian cells canrespond to extracellular dsRNA, and RNAi can act systemically; thereforea specific transport mechanism for dsRNA may exist (see, e.g., Asher etal. (1969) Nature 223:715-717; WO01/36646; WO01/68836). Consequently,injection of dsRNA into one tissue can inhibit gene function in cellsthroughout the animal. Thus, dsRNA may be administered extracellularlyinto a cavity, interstitial space, into the circulation of a mammal,introduced orally, or may be introduced by bathing an organism in asolution containing RNA. Methods for oral introduction include directmixing of the RNA with food of the organism, as well as engineeredapproaches in which a species that is used as food is engineered toexpress the RNA, then fed to the organism to be affected. For example,food bacteria, such as Lactococcus lactis, may be transformed to producethe dsRNA (see, e.g., WO97/17117, WO97/14806). Methods of injectioninclude injection into vascular or extravascular circulation, the bloodor lymph systems and the cerebrospinal fluid are sites where the RNA maybe injected.

Drosophila cells are particularly well-suited for RNAi-based alterationof gene expression. Many Drosophila cell lines have been established andcan be biochemically characterized for use in studying various cellularprocesses. Drosophila cell lines that are known to respond to dsRNAs byablating expression of the target protein include S2, KC, BG2-C6, andShi cells. Many signal transduction pathways and other cellularprocesses have been highly conserved from Drosophila to mammals, makingit possible to study complex biochemical problems in a geneticallytractable model organism. Importantly, results obtained from the cellculture studies can be confirmed in the whole organism, becauseDrosophila is very amenable to RNAi analyses at the organismal level.The use of dsRNA in Drosophila cell culture to silence expression ofspecific genes is technically simple, efficacious, and highlyreproducible. The dsRNAs are efficiently internalized by the cells,thereby circumventing the problems generated by variable transfectionefficiencies. Also, the gene silencing effect can be sustained throughmany cell divisions.

Compared to antisense technology, RNAi has been reported to achievegreater than 95% reduction in gene product. This effect can bemanifested over a period of 6-7 days, thus allowing for many data pointsand repetition of the assay over time (Caplen et al. (2000) Gene252:95-105).

(c) Gene Knock Out or Deletion

Direct gene “knock-out” procedures may also be used to alter theexpression of a gene in a cell. In these methods, homologousrecombination between DNA in a cell and heterologous nucleic acidintroduced into the cell results in elimination of a targeted gene fromthe genome or alteration of the gene such that it does not producefunctional protein. Methods of designing nucleic acid constructs for usein targeted gene disruption or deletion are well known in the art (see,e.g., Capecchi (1989) Science 244:1288; Capecchi et al. (1990) Nature344:105; Koller et al. (1990) Science 248:1227).

(d) Gene Insertion

Transfection methods may be used to introduce a gene, or portionthereof, into a host cell. The nucleic acid(s) transferred into the hostcell may encode a wild-type or altered protein or a domain, derivative,fragment or homolog thereof. Transfer of nucleic acid(s) into a hostcell can be accomplished by a variety of procedures. Such proceduresinclude, but are not limited to, direct uptake using calcium phosphate(CaPO₄; see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A.76:1373-1376), polyethylene glycol (PEG)-mediated DNA uptake,electroporation, lipofection (see, e.g., Strauss (1996) Meth. Mol. Biol.54:307-327), microcell fusion (see, e.g., Lambert (1991) Proc. Natl.Acad. Sci. U.S.A. 88:5907-5911; U.S. Pat. No. 5,396,767, Sawford et al.(1987) Somatic Cell Mol. Genet. 13:279-284; Dhar et al. (1984) SomaticCell Mol. Genet. 10:547-559; and McNeill-Killary et al. (1995) Meth.Enzymol. 254:133-152), lipid-mediated carrier systems (see, e.g., Teifelet al. (1995) Biotechniques 19:79-80; Albrecht et al. (1996) Ann.Hematol. 72:73-79, Holmen et al. (1995) In vitro Cell Dev. Biol. Anim.31:347-351; Remy et al. (1994) Bioconjug. Chem. 5:647-654; Le Bolch etal. (1995) Tetrahedron Lett. 3:6681-6684; Loeffler et al. (1993) Meth.Enzymol. 217:599-618), liposome-mediated delivery (see, e.g., Philip etal. (1993) J. Biol. Chem. 268:16087-16090 and Aksentijevich et al.(1996) Hum. Gen. Ther. 7:1111-1122), adenovirus infection (see, e.g.,Ragot et al. (1993) Nature 361:647-650), retroviral transduction (see,e.g., Cochlovius et al. (1998) Cancer Immunol. Immunother. 46:61-66,Bunnell et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:7739 and Finer etal. (1994) Blood 83:43), electroporation (see, e.g., Hughes et al.(1996) J. Biol. Chem. 271:5369-5377 and Cron et al. (1997) J. ImmunolMeth. 205:145-150), particle bombardment (see, e.g., Yang et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87:9568-9572), direct local injection ofthe DNA (for in vivo transfer of DNA) (see, e.g., Wolff et al. (1990)Science 247:1465-1468; and Zhu et al. (1993) Science 261:209-211),antibody-based methods (Mannino and Gould-Fogerite (1988) Biotechniques6:682-690), retroviruses (Roux et al. (1989) Proc. Natl. Acad. Sci.U.S.A. 86:9079-9083), and antifection (see, e.g., Hirsch et al. (1993)Transpl. Proc. 25:138-139 and Poncet et al. (1996) Gene Therapy3:731-738).

The nucleic acid encoding a molecule (e.g., protein) of interest can beoperably linked to elements that facilitate expression of the nucleicacid in host cells. Such elements include promoters, enhancers andterminators that are functional in the recipient host cell and are knownto those of skill in the art.

For transfer of nucleic acid of interest into cells, the nucleic acidmay be contained within a vector. Any vector known in the art fortransfer and expression of nucleic acids in cells may be used, includingplasmids, cosmids and artificial chromosomes. For simultaneousco-transfection, nucleic acid encoding more than one molecule (e.g.,protein) of interest may be contained on separate vectors or on the samevector in which they can be operably linked to elements that facilitateexpression of the nucleic acids in host cells. Multiple sequences, suchas nucleic acids expressing multiple elements in a calcium flux pathway,contained on the same vector may be controlled either by a singlepromoter or by multiple promoters. In a specific embodiment, thepromoter is not native to the gene(s) expressing the protein(s).

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a nucleic acid of interest and appropriatetranscriptional/translational control signals and/or other proteincoding sequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).

d. Determining Effects of Alteration of Gene Expression on IntracellularCalcium

Following and/or simultaneously with the alteration of gene expressionin a cell, the cell (or portion thereof) is analyzed to evaluate theeffect, if any, on intracellular calcium. Intracellular calcium may beevaluated in any of a number of ways, including any of the methodsdescribed herein or known in the art. For example, intracellular calciumcan be evaluated by assessment of cytosolic or intracellular calciumstore or organelle calcium levels and/or fluxes or by assessment ofcalcium movement into or out of the cell following reduction, alterationor elimination of the expression of a gene encoding a candidate or testmolecule. Cells can be exposed to conditions (e.g., intracellular and/orextracellular calcium buffering, including use of calcium chelators, andexposure to agents that activate, inhibit or otherwise modulate variouscation entry/flux processes) that facilitate assessment of intracellularcalcium. In particular embodiments of the methods provided herein foridentifying intracellular calcium-modulating proteins, cytosolic calciumbuffering, calcium store, organelle or resting cytosolic calcium levels,movement of calcium into, out of or within a calcium store orintracellular organelle and/or store-operated calcium entry areevaluated during and/or following alteration of gene expression. Forexample, calcium levels and/or calcium release from an intracellularcalcium store (e.g., endoplasmic reticulum) can directly be assessedusing mag-fura 2, endoplasmic reticulum-targeted aequorin or cameleons.One method for indirect assessment of calcium levels or release ismonitoring cytosolic calcium levels (for example, usingfluorescence-based methods) after exposing a cell to an agent thateffects calcium release (e.g., non-physiological activators such asthapsigargin, and physiological activators such as IP₃) from theorganelle in the absence of extracellular calcium.

Resting cytosolic calcium levels, intracellular calcium store ororganelle calcium levels and cation movement may be assessed using anyof the methods described herein or known in the art (see, e.g.,descriptions herein of calcium sensitive indicator-based measurements,such as fluo-3, mag-fura 2 and ER-targeted aequorin, labelled calcium(such as ⁴⁵Ca²⁺)-based measurements, and electrophysiologicalmeasurements). Particular aspects of ion flux that may be assessedinclude, but are not limited to, a reduction (including elimination) orincrease in the amount of ion flux, altered biophysical properties ofthe ion current, and altered sensitivities of the flux to activators orinhibitors of calcium flux processes, such as, for example,store-operated calcium entry.

A protein, e.g., an ion transport protein, that is involved in,participates in and/or provides for store-operated calcium entry, and/ornucleic acid encoding such a protein, can be identified by a cell inwhich reduction, alteration or elimination of the expression of a geneis accompanied by an alteration, e.g., reduction, elimination, increaseor other modification, of store-operated calcium entry in the cell. Aprotein, e.g., an ion transport protein, involved in maintenance ofresting cytosolic calcium levels, and/or nucleic acid encoding such aprotein, can be identified by a cell in which reduction, alteration orelimination of the expression of a gene is accompanied by an alteration,e.g., increase, decrease or other modification, of resting cytosoliccalcium levels. A protein, e.g., an ion transport protein, that isinvolved in, participates in, or provides for modulation of calciumlevels in or movement of calcium into, out of or within an intracellularcalcium store or organelle (and/or nucleic acid encoding such a protein)can be identified by a cell in which reduction, alteration orelimination of the expression of a gene is accompanied by an alteration,e.g., increase, decrease, reduction, elimination or other modification,of the calcium level of an intracellular calcium store or organelle orof movement of cations (or other charge carrier) into, out of or withinan intracellular calcium store or organelle.

3. Assays and Tools for Use with Screening Methods

A wide variety of assay methods can be used with thescreening/identification methods described herein. Any assays thatdetect, monitor or measure an effect on intracellular calcium, includingcalcium entry-mediated events can be used. Such assays include, but arenot limited to, assays monitoring, measuring and/or detectingintracellular calcium levels, modulation of calcium levels, and movementof calcium into, out of or within cells and intracellular organelles.Assays can also include monitoring, measuring and/or detecting calciumentry-mediated events and molecules involved in calcium entry-mediatedevents such as, but not limited to, signal transduction molecules,transcription factors, secreted molecules and other molecules that areaffected by changes in calcium homeostasis

a. Cells

In embodiments of the screening/identification methods that involve theuse of cells, any cell that can be evaluated for intracellular calciumcan be used. A wide variety of cell types for such assays are available.Exemplary cells and assays are described herein. In a particularembodiment, the cell is one in which store-operated calcium entry occursor that can be manipulated such that store-operated calcium entry occursin the cell. In particular embodiments, the cell contains one or moreproteins involved in modulating intracellular calcium (and, inparticular, is involved in, participates in and/or provides forstore-operated calcium entry, movement of calcium into, out of or withinan intracellular organelle or calcium store, modulation of calciumlevels in an intracellular organelle or calcium store (e.g., endoplasmicreticulum) and/or calcium buffering), such as those provided herein. Inparticular embodiments, the protein(s) is (or is substantiallyhomologous to) one of the proteins listed in Table 3. The protein canbe, for example, a STIM or STIM-like protein (including a STIM1, STIM2,DSTIM and CSTIM protein). In one embodiment, the protein is a STIM1protein, for example, a mammalian, such as human or rodent, STIM1protein. The cell may endogenously express the protein(s) orrecombinantly express the protein(s), e.g., through introduction ofheterologous nucleic acid encoding the protein(s) into the cell usingmethods known in the art and described herein.

In particular embodiments, the cell is a recombinant cell wherein atleast some of the protein(s) are encoded by nucleic acid that isheterologous to the cell. The cell can be a recombinant cell thatexpresses at least some of the protein(s) as heterologous protein(s).Such cells may overexpress the protein(s), e.g., relative to any levelof expression of the protein(s) in the same cell which has not betreated so as to introduce heterologous nucleic acid encoding theprotein(s). For example, a recombinant cell may be one that endogenouslyexpresses the protein(s) and that also has been transfected withadditional copies of nucleic acid encoding the protein(s). In aparticular example, the host cell used in generating the recombinantcell may be one that endogenously expresses little to no store-operatedcalcium entry activity (e.g., CHO-K1 cells that do not exhibit a currentwith biophysical properties characteristic of a store-operated calciumentry current), or a host cell in which endogenous store-operatedcalcium entry activity has been reduced or eliminated (e.g., throughgene knock-out or silencing, such as RNA interference, methods or byinhibition with an agent that does not inhibit store-operated calciumentry activity of the heterologous protein(s)).

Cells or less differentiated precursor cells having an endogenousprotein involved in modulating intracellular calcium can be used. Cellsor less differentiated precursor cells may be recombinant cells stablyor transiently transfected with intracellular calcium-modulatingprotein(s) in vitro or in an organism. In vitro transfection is followedby cell expansion through culturing prior to use.

Cells for use in the methods may be of any species. In one embodiment,the cells can be eukaryotic cells. In a particular embodiment, the cellscan be yeast, insect (e.g., Drosophila or Anopheles), or mammaliancells. Mammalian cells include, but are not limited to, rodent (e.g.,mouse, rat and hamster), primate, monkey, dog, bovine, rabbit and humancells. A variety of cell types can be used in the methods, including,for example, neuronal, nervous system, brain, immune system cells, e.g.,T lymphocytes and B cells, primary cells, blood and hematopoietic cells,stromal cells, myeloid cells, lymphoid cells, and a variety of tumor andcancer cells. Particular cells include Drosophila Schneider 2 or S2cells, human embryonic kidney (HEK293) cells, rat basophilic leukemia(RBL-2H3) cells, Jurkat cells, epithelial cells, rhabdomyosarcoma cells,rhabdoid cells, retinoblastoma cells, neuroepithelioma cells,neuroblastoma cells, osteosarcoma cells, fibroblasts, bone marrow stromacells, erythroleukemia cells and lymphoblast cells. Other cell linesinclude HEK 293 and 293T, CHO (including CHO-K1), LTK-, N2A, H6, andHGB. Many such cells and cell lines are available through celldepositories such as, for example, the American Type Culture Collection(ATCC, Manassas, Va.). Primary cells can be obtained by isolation fromtissue sources. For example, dorsal root ganglion cells can be isolatedby dissection from the spinal cord of rats using procedures known in theart. The generation, maintenance and use of such cells and cell lines iswell known. The host cell for generation of a recombinant cell may be aless differentiated precursor cell, and may be one that is readilystably or transiently transfected.

In particular embodiments, neuronal, neuroendocrine, brain, or nervoussystem (e.g., CNS) or tissue-derived cells can be used in the method forscreening for or identifying an agent that modulates intracellularcalcium. Such a cell can be one that contains (endogenously and/or asheterologous components, e.g., recombinantly) one or more proteinsinvolved in modulating intracellular calcium (as described above,including proteins listed in Table 3 or substantially homologous toproteins listed in Table 3). Cells from a known cell line can be used,such as neuroblastoma SH-SY5Y cells, pheochromocytoma PC12 cells,neuroblastoma SK-N-BE(2)C or SK-N-SH cells, human SK-N-MCneuroepithelioma cells, SMS-KCNR cells, human LAN-5 neuroblastoma cells,human GI-CA-N neuroblastoma cells, human GOTO neuroblastoma cells, mouseNeuro 2a (N2A) neuroblastoma cells and/or human IMR 32 neuroblastomacells. Primary cells, e.g., dorsal root ganglion and other primaryneuronal or CNS-derived cells, can also be used in the methods.

In another particular embodiment, myeloid cells and cell lines may beused in the methods for screening for or identifying an agent thatmodulates intracellular calcium. Such a cell can be one that contains(endogenously and/or as heterologous components, e.g., recombinantly)one or more proteins involved in modulating intracellular calcium (asdescribed above, including proteins listed in Table 3 or substantiallyhomologous to proteins listed in Table 3). Such cells include, but arenot limited to, chronic myeloid leukemia cells (e.g., human K562 cells),promyelocytic leukemia cells (e.g., HL60 cells) and histiocytic lymphomacells (e.g., U937 cells).

In one embodiment, epithelial-type cells and cell lines may be used inthe methods of screening for or identifying agents that modulateintracellular calcium. Such a cell can be one that contains(endogenously and/or as heterologous components, e.g., recombinantly)one or more proteins involved in modulating intracellular calcium (asdescribed above, including proteins listed in Table 3 or substantiallyhomologous to proteins listed in Table 3). Such cells include, but arenot limited to, rhabdoid tumor cells (e.g., human kidney rhabdoid tumorcells, such as G401 cells) and rhabdomyosarcoma cells (e.g., humanmuscle rhabdomyosarcoma cells, such as A204 cells).

In another embodiment, lymphoid cells and cell lines may be used in themethods of identifying agents that modulate intracellular calcium. Sucha cell can be one that contains (endogenously and/or as heterologouscomponents, e.g., recombinantly) one or more proteins involved inmodulating intracellular calcium (as described above, including proteinslisted in Table 3 or substantially homologous to proteins listed inTable 3). Such cells include, but are not limited to, Burkitt's lymphomacells (e.g., CA46 cells), B-cells (e.g., NALM6), acute lymphoblasticleukemia cells (e.g., MOLT4cells), T cells (e.g. Jurkat cells) and earlyT-ALL (e.g., DU528) cells.

The choice of a cell for use in practicing any of the methods forscreening for or identifying an agent involved in modulatingintracellular calcium can involve several considerations, including, forexample, a particular protein that is being used in the method and aparticular aspect or activity of intracellular calcium modulation thatis being monitored or assessed in the method. For example, differentcells (e.g., different cell types or cells of the same type fromdifferent species of organisms) may have different sets of molecules,including proteins and ion transport proteins, that participate invarious aspects (e.g., store-operated calcium entry, receptor-mediatedcalcium movement, second messenger-operated calcium movement, calciumuptake, storage and/or release from intracellular compartments orstores, calcium buffering) of intracellular calcium modulation.Accordingly, a cell used in the methods can be selected to be one thatprovides (e.g., endogenously and/or recombinantly) a cellularenvironment conducive for functioning of any particular protein(s) (suchas those provided herein and described above, including proteins listedin or substantially homologous to protein listed in Table 3) being usedin the method or supportive of any particular activity in intracellularcalcium modulation that is being specifically monitored or assessed inthe methods. A cell may also be selected based on relative amounts ofparticular proteins expressed by the cell. For example, in someembodiments of the methods, it may be desirable to utilize a cell thatcontains more STIM1 than STIM2 protein (e.g., a PC12 cell).Additionally, some cells may be more amenable to various manipulationsthat may be a part of some embodiments of the methods, e.g., recombinantexpression of proteins, growth in culture, electrophysiologicalanalysis, etc. Cells that are particularly suitable for specificembodiments of the methods can be determined empirically by those ofskill in the art.

For example, particular embodiments of the methods for screening for oridentifying agents that modulate intracellular calcium include a step ofmonitoring or assessing the effect of a test agent on store-operatedcalcium entry. These methods can involve the use of cells for assessingeffects on store-operated calcium entry. Cells typically used in suchmethods exhibit store-operated calcium entry either naturally or throughmanipulation of the cells. Cells that endogenously exhibitstore-operated calcium entry include some excitable cells and mostnon-excitable cells and can be identified using methods described hereinand/or known in the art. Any method of determining the occurrence ofcalcium entry into a cell that distinguishes store-operated calciumentry from other types of calcium influx (e.g., entry throughvoltage-gated calcium channels or other channels that are not dependenton depletion of calcium stores) can be used to determine if a cellexhibits store-operated calcium entry. It may also be possible tomanipulate some cells that exhibit low levels or undetectable levels ofstore-operated calcium entry such that they produce store-operatedcalcium entry activity, or exhibit more readily detectable levels ofstore-operated calcium entry activity. For example, transfer andexpression of nucleic acids encoding one or more proteins (or portionsthereof) described herein as proteins involved in intracellular calciummodulation (including STIM, STIM-like proteins and proteins listed in orproteins substantially homologous to proteins listed in Table 3) intosuch cells may result in store-operated calcium entry activity orincreased stored-operated calcium entry activity in the recombinantcells. Such cells can be evaluated to determine the presence or level ofstore-operated calcium entry activity using procedures described hereinand/or known in the art.

In embodiments of the screening/identification methods that include astep of monitoring or assessing the effect of a test agent onstore-operated calcium entry, it may be desirable to utilize a cell thatcontains components of signaling and messenger systems that can effectrelease of calcium from intracellular stores. For example, cellscontaining components of receptor-mediated phospholipase C (PLC)activation systems can be used for physiological activation (viageneration of IP₃) of store depletion to facilitate monitoring ofstore-operated calcium entry. Receptor-mediated PLC activation occursthrough distinct coupling mechanisms: PLC-β activation by Gprotein-coupled receptors (GPCRs) and PLC-γ activation by tyrosinekinase receptors and nonreceptor tyrosine kinases. Components ofreceptor-mediated PLC activation systems include GPCRs (e.g., muscarinicacetylcholine receptors, purinergic receptors, β-adrenergic receptorsand serotonin receptors), tyrosine kinase receptors (e.g., growth factorreceptors, including EGF, FGF, PDGF and NGF receptors), nonreceptortyrosine kinases (e.g., Src, Syk and Tec which can be activated byantigen and Ig receptors, such as the T-cell antigen and B-cell antigenreceptors) and G proteins. Thus, cells containing a receptor-mediatedPLC-activation system can be monitored or assessed for store-operatedcalcium entry upon agonist activation of one or more receptors known toparticipate in the system. A cell can contain an endogenousreceptor-mediated PLC-activation system or can be manipulated (e.g., byintroduction and expression of heterologous nucleic) to express such asystem. For example, store-operated calcium entry can be monitored orassessed in PC12 cells upon activation of purinergic P2Y receptors(e.g., by UTP), in cultured hippocampal neurons, human neuroblastomacells (e.g., SH-SY5Y cells) and human embryonic kidney (e.g., HEK 293)cells upon activation with the cholinergic agonists carbachol ormethacholine (see e.g., Bouron (2000) FEBS Lett 470:269-272) and in DT40chicken B lymphocyte cells (which express PLC-β) and Drosophila S2 cellsupon activation of G protein-coupled muscarinic (e.g., M5 or DrosophilaM1) receptors (e.g., by carbachol) recombinantly expressed throughtransfection of the cells with nucleic acid encoding a muscarinicreceptor (see, e.g., Millar et al. (1995) J. Exp. Biol. 198:1843-1850;Yagodin et al. (1998) Cell Calcium 23:219-228; Yagodin et al. (1999)Cell Calcium 25:429-438; and Patterson et al. (2002) Cell 111:1-20).Cells containing receptor-mediated PLC-activation systems, and reagents(e.g., nucleic acids encoding receptors, such as muscarinicacetylcholine receptors, and other elements of receptor-mediatedPLC-activation systems) and methods for use in generating such systems,are known in the art.

b. Monitoring or Assessing Effects on Intracellular Calcium

In monitoring or assessing the effect of a test agent or molecule onintracellular calcium in any of the screening/identification methodsprovided herein, a direct or indirect evaluation or measurement ofcellular (including cytosolic and intracellular organelle orcompartment) calcium and/or movement of ions into, within or out of acell, organelle, calcium store or portions thereof (e.g., a membrane)can be conducted. A variety of methods are described herein and/or knownin the art for evaluating calcium levels and ion movements or flux. Theparticular method used and the conditions employed can depend on whethera particular aspect of intracellular calcium is being monitored orassessed. For example, as described herein, reagents and conditions areknown, and can be used, for specifically evaluating store-operatedcalcium entry, resting cytosolic calcium levels, calcium buffering andcalcium levels and uptake by or release from intracellular organellesand calcium stores. The effect of a test agent or molecule onintracellular calcium can be monitored or assessed using, for example, acell, an intracellular organelle or calcium storage compartment, amembrane (including, e.g., a detached membrane patch or a lipid bilayer)or a cell-free assay system (e.g., outside-out membrane vesicle).

Generally, monitoring or assessing the effect of a test agent ormolecule on intracellular calcium involves contacting a test agent with(1) a protein (and/or nucleic acid, or portion(s) thereof, encoding aprotein) involved in modulating intracellular calcium (in particular, aprotein provided herein) and/or (2) a cell, or portion(s) thereof (e.g.,a membrane or intracellular structure or organelle) or cell-free systemthat may or may not contain a protein (and/or nucleic acid, orportion(s) thereof, encoding a protein) involved in modulatingintracellular calcium. A cell can be one that exhibits one or moreaspects of intracellular modulation, such as, for example,store-operated calcium entry. Before, during and/or after the contactingof test agent or molecule, a direct or indirect assessment ofintracellular calcium can be made. An indirect assessment can be, forexample, evaluation or measurement of current through an ion transportprotein (e.g., a store-operated calcium channel), or detection of theexpression of a reporter protein operably linked to a calcium-sensitivepromoter. A direct assessment can be, for example, evaluation ormeasurement of intracellular (e.g., cytosolic or intracellularorganelle) calcium.

The assessment of intracellular calcium is made in such a way as to beable to determine an effect of an agent on intracellular calcium.Typically, this involves comparison of intracellular calcium in thepresence of a test agent or molecule with a control for intracellularcalcium. For example, one control is a comparison of intracellularcalcium in the presence and absence of the test agent or molecule or inthe presence of varying amounts of a test agent or molecule. Thus, onemethod for monitoring or assessing an effect on intracellular calciuminvolves comparing intracellular calcium before and after contacting atest agent or molecule with a test cell or portion thereof (e.g., acell, or portion thereof, containing a protein (and/or nucleic acidencoding a protein) involved in modulating intracellular calcium), orcomparing intracellular calcium with respect to a test cell (or portionthereof) that has been contacted with or exposed to test agent and withrespect to a cell (or portion thereof) that has not been contacted withor exposed to test agent or molecule (i.e., a control cell or system).Generally, the control cell (or portion thereof) is substantiallyidentical to, if not the same as, the test cell (or portion thereof),except it is the cell (or portion thereof) in the absence of test agent.A difference in intracellular calcium of a test cell (or portionthereof) in the presence and absence of test agent or molecule or adifference in intracellular calcium of a test cell (or portion thereof)and a control cell (or portion thereof) indicates that the agent ormolecule is one that modulates intracellular calcium.

In embodiments of the screening/identification methods that involveexposing a test cell (or portion thereof) containing a protein (orportion thereof) involved in modulating intracellular calcium to a testagent, another type of control cell (or portion thereof) can be used inthe methods. Such a control cell (or portion thereof) is substantiallysimilar to the test cell but contains a different amount or level of theparticular protein (or activity of the particular protein) involved inmodulating intracellular calcium than the amount or level of the protein(or activity of the protein) contained in the test cell. For example,the control cell (or portion thereof) can contain more of the protein(or greater activity of the protein) involved in modulatingintracellular calcium or less of the protein (or reduced activity of theprotein) compared to the amount of the protein (or the activity level ofthe protein) contained in the test cell (or portion thereof).

Such control cells (or portions thereof) can be generated in a number ofways. For example, a substantially similar control cell of this typecould be generated by altering the expression of a particular proteininvolved in modulating intracellular calcium in a test cell usingmethods described herein and/or known in the art. Expression-reducing(or eliminating) techniques include RNA interference, antisense RNA andgene knock-out methods. Expression-increasing techniques includeintroduction of additional copies of nucleic acid encoding theparticular protein into a test cell (e.g., over-expression of theparticular protein). A control cell generated by altering expression ofa protein in a test cell is substantially similar to the test cellexcept that it contains more or less of the protein (or proteinactivity) than the test cell. In another example, a control cell canalso be one that is not manipulated in the same way as a test cell. Forexample, if the test cell containing the protein involved inintracellular calcium modulation is a recombinant cell generated bytransfer of nucleic acid encoding the protein into a host cell, then onepossible control cell is a host cell that has not been transfected withnucleic acid encoding the protein. If the host cell is one that does notcontain nucleic acid encoding the protein, then the control cell issubstantially similar to the test cell except that it lacks theparticular protein involved in modulating intracellular calcium. If thehost cell is one that does express some level of the particular protein,then the control cell is substantially similar to the test cell exceptthat it contains less nucleic acid encoding the particular protein (andless of the particular protein) than the test cell. Thus, a control cellmay contain, e.g., endogenously, the particular protein involved inmodulating intracellular calcium, in which case the test cell wouldcontain higher levels of (or overexpress) the particular protein.

When a control cell (or portion thereof) used in the method is one thatis substantially similar to a test cell but contains a different amountor level of the particular protein (or activity of the particularprotein) involved in modulating intracellular calcium than the amount orlevel of the protein (or activity of the protein) contained in the testcell, an effect of test agent on intracellular calcium can be monitoredor assessed by comparing intracellular calcium of the test and controlcell in the presence of test agent. This type of control comparison isparticularly of use when it is desired to identify an agent thatspecifically modulates intracellular calcium via an effect on, ormodulation of, a particular protein (and/or nucleic acid, or portion(s)thereof, encoding a particular protein). Thus, for example, if there isno detectable or substantial difference in intracellular calcium in thetest and control cells in the presence of the agent, it is not likelythat the agent has an effect on intracellular calcium that isspecifically mediated via the particular protein (or nucleic acidencoding the protein). If there is a detectable or substantialdifference in intracellular calcium in the test and control cells in thepresence of the test agent, then the test agent may be a candidate agentthat specifically modulates intracellular calcium via an effect on ormodulation of the particular protein. A candidate agent can be subjectedto further control assays to compare intracellular calcium in test cellsin the presence and absence of test agent and/or to compareintracellular calcium in control cells in the presence and absence oftest agent, which can aid in determination of whether a candidate agentis an agent that modulates intracellular calcium. For example, anydifference in intracellular calcium in a control cell in the presenceand absence of test agent can be compared to any difference inintracellular calcium in a test cell in the presence and absence of testagent. If the relative differences in intracellular calcium for the testand control cells are similar or do not differ substantially, then itmay not be likely that the agent has an effect on intracellular calciumthat is mediated via the particular protein.

An assessment of intracellular calcium conducted to monitor the effectof test agent or molecule on intracellular calcium can be made under avariety of conditions. Conditions can be selected to evaluate the effectof test agent on a specific aspect of intracellular calcium. Forexample, as described herein, reagents and conditions are known, and canbe used, for specifically evaluating store-operated calcium entry,resting cytosolic calcium levels, calcium buffering, and calcium levelsof and calcium uptake by or release from intracellular organelles.Resting cytosolic calcium levels, intracellular organelle calcium levelsand cation movement may be assessed using any of the methods describedherein or known in the art (see, e.g., descriptions herein ofcalcium-sensitive indicator-based measurements, such as fluo-3, mag-fura2 and ER-targeted aequorin, labelled calcium (such as ⁴⁵Ca²⁺)-basedmeasurements, and electrophysiological measurements). Particular aspectsof ion flux that may be assessed include, but are not limited to, areduction (including elimination) or increase in the amount of ion flux,altered biophysical properties of the ion current, and alteredsensitivities of the flux to activators or inhibitors of calcium fluxprocesses, such as, for example, store-operated calcium entry. Reagentsand conditions for use in specifically evaluating receptor-mediatedcalcium movement and second messenger-operated calcium movement are alsoavailable.

i. Evaluation of Store-Operated Calcium Entry

In particular embodiments of the methods for screening for oridentifying agents and molecules that modulate intracellular calcium,the methods are conducted under conditions that permit store-operatedcalcium entry to occur. Such conditions are described herein and areknown in the art. Test agents can be contacted with a protein and/ornucleic acid encoding, or regulating the expression of, a protein (suchas the proteins and nucleic acids provided herein) involved inmodulating intracellular calcium and/or a cell (or portion thereof)containing such a protein (or nucleic acid) under these appropriateconditions.

For example, in one method for detecting or monitoring store-operatedtransport of calcium across the plasma membrane, cells may be treated toreduce the calcium levels of intracellular calcium stores and thenanalyzed for evidence of ion (particularly cation, e.g., calcium) influxin response thereto. Techniques for reducing calcium levels ofintracellular stores and for analyzing cells for evidence of ion(particularly cation, e.g., calcium) influx are known in the art anddescribed herein.

In other methods, diffusible signals may be used to activatestore-operated calcium entry in methods of detecting and monitoring thesame. One such signal is referred to as calcium influx factor (CIF)(see, e.g., Randriamampita and Tsien (1993) Nature 364:809-814; Parekhet al. (1993) Nature 364:814-818; Csutora et al. (1999) Proc. Natl.Acad. Sci. U.S.A. 96:121-126), which may be a small (˜<500 D)phosphate-containing anion. A CIF activity from thapsigargin-treatedJurkat cells, as well as a similar activity from calcium pump-deficientyeast, can activate calcium influx in Xenopus oocytes and in Jurkatcells. When included in the patch pipette during whole-cell patch clampof Jurkat cells, the extracts activate an inward current resemblingI_(CRAC).

In other methods, electrophysiological analysis of currents across acell-detached plasma membrane patch or an outside-out membrane vesiclemay be used to detect or monitor store-operated channel currents (e.g.,I_(CRAC))

(a) Reduction of Calcium Levels of Intracellular Stores

A variety of treatments may be used to reduce calcium levels inintracellular calcium stores. Generally, the treatments can be viewed aseither an active, direct reduction in calcium levels, such as by removalof free calcium from the stores (i.e., “active” depletion), or a passivereduction in calcium levels, such as by leak of calcium from the storeseither by a reduction in the availability of free calcium for filling orreplenishing the stores or by preventing filling or replenishing ofstores (i.e., “passive” depletion).

(1) Passive Depletion

One method of reducing the availability of calcium for the internalcalcium stores is to decrease the calcium concentration of theextracellular medium and/or cytoplasm. Calcium concentrations of thesefluids can be decreased using cation, and particularly calcium,chelators including, but not limited to EGTA and1,2-bis(2-amino-phenoxy)ethane-N,N,N,N′-tetraacetic acid (BAPTA). Forexample, cells may be equilibrated in 10 mM external calcium with strongbuffering of cytosolic calcium, for example, through dialysis with 10 mMEGTA. Alternatively, reduction of external calcium may also depleteintracellular calcium stores in many types of cells. For example, cellsmay be incubated in nominally free calcium solution, e.g., 10 μMexternal calcium, or essentially calcium-free solution, e.g., ˜1 nMexternal calcium with strongly (e.g., 10 mM EGTA) buffered cytosoliccalcium.

A membrane-permeant cation chelator that can chelate calcium withininternal stores may also be used to reduce free calcium levels of thestores. One such chelator is N,N,N,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), which, in its uncomplexed form, diffuses across cellmembranes (see, e.g., Hofer et al. (1998) J. Cell Biol. 140:325-334).Because this multivalent cation chelator has a low affinity for calcium,it should not significantly influence calcium levels in the cytoplasm orother cell compartments where the steady-state calcium concentration isin the nanomolar or low micromolar range. In cell compartments where thecalcium concentration is comparable to its Kd, such as, for example, theendoplasmic reticulum, TPEN should bind calcium to rapidly reduce freecalcium levels. Removal of TPEN from the cell medium should provide forincreases in free calcium levels in such cell compartments due to rapidunbinding of the chelator from calcium ions and diffusion of the freeform of TPEN from the compartment. Thus, TPEN may be used to reversiblymanipulate store calcium levels without interfering with other aspectsof calcium homeostasis.

Reduction of calcium in intracellular calcium stores can also beaccomplished by application of an agent that blocks endoplasmicreticulum calcium ATPase pumps (SERCAs), thereby reducing or preventingrefilling of the endoplasmic reticulum with calcium and providing forleak of calcium from the ER into the cytoplasm resulting in a reductionof ER free calcium concentration. For example, ER free calciumconcentration may decrease from about 500 μM to about 50-100 μM as hasbeen observed in HEK293 cells (Yu and Hinkle (2000) J. Biol. Chem.275:23648-23653). Such agents include, but are not limited to,thapsigargin, cyclopiazonic acid (CPA), and di-tert-butyl-hydroquinone(tBHQ). Agents such as these are referred to as nonphysiologicalactivators of calcium store depletion.

(2) Active Depletion

Active reduction of calcium levels in intracellular calcium stores canbe experimentally implemented in a number of ways. For example, exposureof endoplasmic reticulum inositol-1,4,5-triphosphate (IP₃) receptors toIP₃ or derivatives or analogs thereof provides for release of calciumfrom this calcium store and serves to reduce calcium levels therein. IP₃or derivatives or analogs thereof can be provided to the endoplasmicreticulum through direct intracellular application, through applicationto the plasma membrane (using membrane-permeable derivatives of IP₃) orby contacting cells with or exposing cells to an agent that activatesthe phosphoinositide cascade to generate IP₃. Such activating agentsinclude agonists of plasma membrane receptors linked to activation ofPLC and agents that activate PLC downstream of the plasma membrane inthe signaling cascade. Examples of G protein-coupled receptor agonistsinclude histamine, muscarine, carbachol, substance P, bradykinin,serotonin, uridine triphosphate (UTP) and glutamate. Additionally,inhibition of catabolic enzymes involved in degradation of IP₃ can serveto provide IP₃ for interaction with its receptor. IP₃ and agents thatactivate the phosphoinositide cascade are referred to as physiologicalactivators of calcium store depletion.

Ionophores, e.g., ionomycin (a Ca²⁺/proton ionophore), also may be usedfor active depletion of intracellular calcium stores by embedding in theER membrane and providing a pore-like mechanism for movement of calciumout of the ER.

(b) Analysis of Ion Flux

Many methods for monitoring or detecting store-operated calcium entryinclude a step of detecting and/or analyzing ion flux into a cell oracross a membrane. Techniques for detection and analysis of ion fluxinto and within cells and across membranes are well known in the art.Typically, a cell or membrane can be exposed to conditions that activatestore-operated calcium entry or store-operated calcium channels,including conditions described herein, such as treatment with an agonistthat results in generation of IP₃, internal perfusion with IP₃,treatment with a physiological or non-physiological store-depletionagent e.g., thapsigargin, buffering of cytoplasmic calcium or treatmentwith a CIF, and then analyzed for evidence of movement of ions across amembrane and/or into a cell. Depending on the assay conditions, the ionmay be calcium or other cation, for example sodium or manganese, thatcan be transported via a store-operated channel.

When it is desired to specifically monitor only store-operated calciumentry or ion flux, particular conditions and/or methods can be used inthe analysis. For example, as described herein and in the art,store-operated channel currents (e.g., I_(CRAC)) have distinct featuresthat can be detected through electrophysiological analysis methods andused to distinguish store-operated calcium entry or ion flux. Inaddition, non-physiological activators of store depletion (e.g.,thapsigargin) can be used to specifically activate store-operatedcalcium entry without possible interference by other fluxes (calcium orother ion) that may possibly be induced by physiological activators(e.g., activators of receptor-mediated PLC activation). It may bepossible to detect interfering fluxes (e.g., through receptor-operatedchannels which are not dependent on calcium store depletion foractivation) by using a control cell in which store depletion that wouldoccur upon treatment with a physiological activator is reduced oreliminated by an antagonist of the IP₃ receptor (e.g.2-aminoethoxydiphenylborate (2-APB)). Changes in intracellular calciumor other ion levels, or calcium (or other ion) movement into such acontrol cell (or across a control cell membrane) upon treatment with aphysiological activator would not be reflective of store-operatedchannel activity or store-operated calcium entry.

Ion flux, such as occurs in store-operated calcium entry, can bemeasured electrophysiologically, for example, using patch clamp methods(see, e.g. Hamil et al. (1981) Pflugers Arch. 391:85-100); Hoffman etal. (1999) Nature 397:259-263; and Krause et al. (1999) J. Biol. Chem.274:36957-36962) to record the inward Ca²⁺ (or other ion or chargecarrier) current. The current may be highly selective for Ca²⁺ divalentions, may display negative feedback regulation by Ca²⁺ (see, e.g.Zweifach and Lewis (1995) J. Gen. Physiol. 105:209-226), and may beinhibited by divalent and trivalent metal ions such as Zn²⁺, Ni²⁺, Gd³⁺and La³⁺ (Parekh and Penner (1997) Physiol. Rev. 77: 901-930). Negativefeedback by Ca²⁺ can be eliminated by the inclusion of very highconcentrations of Ca²⁺ buffers in the patch pipette. In the absence ofthese buffers or with buffers of lower capacity, the current may be toosmall to be detected, although Ca²⁺ entry clearly occurs. The currentmay be strongly inwardly rectifying and may lose its property of inwardrectification in the complete absence of divalent cations. It may bepossible to determine the time course of activation of the current insingle cells followed by passive store depletion via patch pipettescontaining Ca²⁺ chelating reagent BAPTA in the whole cell configurationusing Na²⁺ used as the charge carrier (Kerschbaum and Cahalan (1999)Science 283:836).

The electrophysiological measurement/recording of store-operated ionflux through a membrane can provide for the assessment of suchbiophysiological properties as kinetics, voltage dependence and ionicselectivity. The electrophysiological measurement can be performed byusing whole cell patch clamp methods which can allow for the reliableand precise determination of the conditions under which Ca²⁺ influxoccurs. As described by Hofman et al. ((1999) Nature 397:259-263), thepatch clamp technique can be used in whole-cell, cell-attached,inside-out and outside-out mode.

Additionally patch clamp methods can be performed in a perforated-patchwhole cell, configuration. Variations of the patch-clamp technique orother methods for detecting and analyzing ionic activity of cells, whichare routine in the art, can also be used.

Measurement of changes of intracellular ions, such as cations includingCa²⁺, can also can also be performed using fluorescence imaging, such asfluorescence videomicroscopy, digital imaging or ratioimagingtechniques. Measurement of changes in intracellular Ca²⁺ ((Ca²⁺)i) inindividual cells by fluorescence videomicroscopy can be performed usinga digital imaging system, such as, for example, that produced byT.I.L.L. Photonics, or the Attofluor Digital Imaging and Photometryattachment of a Carl Zeis axiovert inverted microscope. Typically, cellscan be grown on coverslips, rinsed and incubated with 5 μM fura2/AM(Molecular Probes) at 37° C. for 30 minutes and then washed with HPSS.The coverslips with the cells are then typically clamped into a circularopen-bottom chamber and mounted onto the stage of a microscope. (Ca²⁺)ican be calculated from fluorescence ratios obtained at 340 nm and 380 nmexcitation wavelengths (Garcia et. al (1994) J. Neurosci. 14:1233-1246).Modified protocols such as those described by Zitt et. al. ((1997) J.Cell Biol. 138:1333-1341) and alternate forms of fluorescence ratioimaging may also be used.

Calcium-sensitive indicators, such as fluo-3 (Catalog No. F-1241,Molecular Probes, Inc., Eugene, Oreg.) and fluo-4, are available asacetoxymethyl esters which are membrane permeable. When theacetoxymethyl ester form of the indicator enters a cell, the ester groupis removed by cytosolic esterases, thereby trapping the free indicatorin the cytosol. Interaction of the free indicator with calcium resultsin increased fluorescence of the indicator; therefore, an increase inthe intracellular Ca²⁺ concentration of cells containing the indicatorcan be expressed directly as an increase in fluorescence.

Additionally, calcium flux in a cell may be monitored using a reportergene expression system. In such a system, the cell in which calciumlevels and fluctuations therein are monitored may contain a reportergene encoding a detectable signal, such as luciferase, which is linkedto a transcription regulatory element, e.g., promoter, that is inducedby calcium-dependent factors.

For example, in conducting one method for screening for or identifyingan agent that modulates intracellular calcium under conditions selectedfor evaluating store-operated calcium entry, intracellular calciumlevels of test cells are monitored over time using a fluorescent calciumindicator (e.g., FLUO-4). Store-operated calcium entry into the cells isdetected as an increase in fluorescence (i.e., increase in intracellularcalcium levels) in response to conditions under which store-operatedcalcium entry occurs. The conditions include addition of astore-depletion agent, e.g., thapsigargin (which inhibits the ER calciumpump and discharges calcium stores) to the media of cell that has beenincubated in Ca²⁺-free buffer, incubation with thapsigargin for about5-15 minutes, addition of test compound (or vehicle control) to themedia and incubation of the cell with test agent for about 5-15 minutes,followed by addition of external calcium to the media to a finalconcentration of about 1.8 mM. By adding thapsigargin to the cell in theabsence of external calcium, it is possible to delineate the transientincrease in intracellular calcium levels due to calcium release fromcalcium stores and the more sustained increase in intracellular calciumlevels due to calcium influx into the cell from the external medium(i.e., store-operated calcium entry through the plasma membrane that isdetected when calcium is added to the medium). Because thefluorescence-based assay allows for essentially continuous monitoring ofintracellular calcium levels during the entire period from prior toaddition of thapsigargin until well after addition of calcium to themedium, not only can “peak” or maximal calcium levels (as well as totalcalcium entry) resulting from store-operated calcium entry be assessedin the presence and absence of test agent, a number of other parametersof the calcium entry process may also be evaluated, as described herein.For example, the kinetics of store-operated calcium entry can beassessed by evaluation of the time required to reach peak intracellularcalcium levels, the up slope and rate constant associated with theincrease in calcium levels, and the decay slope and rate constantassociated with the decrease in calcium levels as store-operated calciumentry discontinues. Any of these parameters can be evaluated andcompared in the presence and absence of test agent to determine whetherthe agent has an effect on store-operated calcium entry, and thus onintracellular calcium. In other embodiments, store-operated calciumentry can be evaluated by, for example, assessing a current across amembrane or into a cell that is characteristic of a store-operatedcalcium entry current (e.g., responsiveness to reduction in calciumlevels of intracellular stores) or assessing transcription of reporterconstruct that includes a calcium-sensitive promoter element. Inparticular embodiments, a test agent is identified as one that producesat least a 50% difference in any aspect or parameter of store-operatedcalcium entry relative to control (e.g., absence of compound, i.e.,vehicle only).

ii. Evaluation of Intracellular Calcium Stores

In particular embodiments of the methods for screening for oridentifying agents that modulate intracellular calcium, the method isconducted under conditions that permit evaluation or monitoring ofintracellular calcium stores, such as, for example, the endoplasmicreticulum. Such conditions are described herein and are known in theart. A cell (or portion thereof, e.g., an intracellular organelle orcalcium store) can be contacted with or exposed to an agent under theseappropriate conditions. The cell can be one, for example, that containsa protein (or nucleic acid encoding, or regulating the expression of, aprotein) involved in modulating intracellular calcium (such as proteinsprovided herein, including STIM and STIM-like proteins and proteinslisted in Table 3).

For example, in one method for detecting or monitoring intracellularcalcium stores, calcium levels and/or calcium uptake by or release fromthe endoplasmic reticulum can directly be assessed using mag-fura 2,endoplasmic reticulum-targeted aequorin or cameleons. One method forindirect assessment of calcium levels in, or calcium release from, anintracellular store involves monitoring or assessing intracellularcalcium levels, such as cytosolic calcium levels and time course ofchanges therein (for example using fluorescence-based methods), afterexposing a cell to an agent that effects calcium release (actively,e.g., IP₃ or caffeine, or passively, e.g., thapsigargin) from thecalcium store or organelle in the absence of extracellular calcium. Theamplitude and rate of the increase in cytosolic calcium levels areindicative of the calcium levels in and calcium release process of theintracellular calcium store.

In particular methods of screening for or identifying an agent thatmodulates intracellular calcium provided herein, the effect of a testagent (which is one that interacts with, binds to and/or modulatesinteractions, activities, levels or any physical, structural or otherproperty of a protein involved in modulating intracellular calcium) onintracellular calcium store calcium level and/or calcium release ismonitored or assessed. In another embodiment, the effect of a test agenton intracellular calcium store calcium level and/or calcium release ismonitored or assessed in a cell that contains a protein involved inmodulating intracellular calcium. In either of these particularembodiments, the protein involved in modulating intracellular calciumcan be one of the proteins described herein above, such as a STIM orSTIM-like protein, or a protein listed in Table 3 or substantiallyhomologous to a protein listed in Table 3. For example, the protein canbe a STIM1 protein, such as a mammalian, and in particular, human, STIM1protein. As described herein, calcium release fromthapsigargin-sensitive calcium stores may be altered in human embryonickidney (HEK293) and neuroblastoma (SH-SY5Y) cells in which STIM1 geneexpression is reduced (as evidenced by decreased mRNA and proteinlevels) by RNA interference methods. This finding suggests STIM1 may beinvolved in regulation of calcium store (e.g., endoplasmic reticulum)calcium levels and/or release of calcium from calcium stores. Thus, inparticular embodiments of the methods for screening for identifying anagent that modulates intracellular calcium, the effect of a test agenton intracellular calcium store calcium level and/or calcium release ismonitored or assessed, wherein the test agent is one that interacts withand/or modulates a STIM1 protein or the test cell is one that contains aSTIM1 protein.

iii. Evaluation of Calcium Buffering

In particular embodiments of the methods for screening for oridentifying agents that modulate intracellular calcium, the method isconducted under conditions that permit evaluation or monitoring ofintracellular (e.g., cytosolic) calcium buffering. Such conditions aredescribed herein and/or are known in the art. A cell can be contactedwith or exposed to a test agent under these appropriate conditions. Thecell can be, for example, one that contains a protein (or nucleic acidencoding, or regulating the expression of, a protein) involved inmodulating intracellular calcium (such as proteins provided herein,including STIM and STIM-like proteins and proteins listed in Table 3).

For example, in one method for detecting or monitoring calciumbuffering, involves monitoring or assessing intracellular calciumlevels, such as cytosolic calcium levels and time course of changestherein (for example using fluorescence-based methods), after exposing acell to an agent that effects calcium release (actively, e.g., IP₃ orcaffeine, or passively, e.g., thapsigargin) from a calcium store ororganelle in the absence of extracellular calcium. The properties orcharacteristics (e.g., rates, kinetics or timing) of the decrease incytosolic calcium levels is indicative of the buffering of calcium(e.g., by uptake of calcium into intracellular organelles or calciumstores and by transfer of calcium from the cytoplasm to theextracellular medium, such as by extrusion from the cell through theaction of the plasma membrane calcium ATPase). Thus, for example,monitoring of or assessing calcium buffering can involve evaluating therate at which cytosolic calcium levels return to basal levels afteractivation of calcium influx into the cytoplasm, the overall time courseof cytosolic calcium level adjustment, timing of the onset of theadjustment of cytosolic calcium levels to return to basal levels afteractivation of calcium influx into the cytoplasm, and the final cytosoliccalcium level (e.g., basal level) attained upon adjustment of calciumlevels after activation of calcium influx.

In particular methods of screening for or identifying an agent thatmodulates intracellular calcium provided herein, the effect of a testagent (which is one that interacts with, binds to and/or modulatesinteractions, activities, levels or any physical, structural or otherproperty of a protein involved in modulating intracellular calcium) oncalcium buffering is monitored or assessed. In another embodiment, theeffect of a test agent on calcium buffering is monitored or assessed ina cell that contains a protein involved in modulating intracellularcalcium. In either of these particular embodiments, the protein involvedin modulating intracellular calcium can be one of the proteins describedherein above, such as a STIM or STIM-like protein, or a protein listedin Table 3 or substantially homologous to a protein listed in Table 3.For example, the protein can be a STIM1 protein, such as a mammalian,and in particular, human, STIM1 protein. As described herein, calciumbuffering capacity is altered in human embryonic kidney (HEK293) cellsin which STIM1 gene expression is reduced (as evidenced by decreasedmRNA and protein levels) by RNA interference methods. For example, therate and kinetics of the adjustment (i.e., decrease) in cytosoliccalcium levels following methylcholine-induced calcium release fromintracellular calcium stores in such cells is altered relative to HEK293cells in which STIM1 expression has not been reduced. This findingindicates involvement of STIM1 in calcium buffering (e.g., by uptake ofcalcium into intracellular organelles or extrusion of calcium to theexterior of the cell). Thus, in particular embodiments of the methodsfor screening for identifying an agent that modulates intracellularcalcium, the effect of a test agent on calcium buffering is monitored orassessed, wherein the test agent is one that interacts with and/ormodulates a STIM1 protein or the test cell is one that contains a STIM1protein.

iv. Evaluation of Resting Cytosolic Calcium Levels

In particular embodiments of the methods for screening for oridentifying an agent that modulates intracellular calcium, the method isconducted under conditions that permit evaluation or monitoring of basalor resting cytosolic calcium levels. Such conditions are describedherein and/or known in the art. A cell can be contacted with or exposeto a test agent under these appropriate conditions. The cell can be one,for example, that contains a protein (or nucleic acid encoding, orregulating the expression of, a protein) involved in modulatingintracellular calcium (such as proteins provided herein, including STIMand STIM-like proteins and proteins listed in Table 3).

For example, resting cytosolic calcium levels can be evaluated using anytechniques for detecting or measuring cytosolic calcium known in the artand/or described herein, including fluorescent calcium indicators. Theevaluation is conducted while a cell is at rest with respect tocytosolic calcium. A cell is at rest with respect to cytosolic calciumwhen it has not been subjected to conditions that result in activationof calcium flux into the cytoplasm from the external medium or calciumrelease from intracellular calcium stores into the cytoplasm.

c. Evaluation of Calcium Entry-Mediated Events

A number of molecules involved in calcium-regulated pathways are known(see for example, FIG. 1 which shows exemplary molecules involved incalcium-entry mediated events in immune cells). Evaluation of moleculesinvolved in calcium-entry mediated events can be used to monitorintracellular calcium, and can be used, for example in screening assaysdescribed herein to monitor the effects of, or identify, test agents andmolecules. Examples of assays include but are not limited to assayswhich detect, or determine the presence, levels, alteration of levels,production, modification (such as phosphorylation anddephosphorylation), translocation, degradation and activity of moleculesinvolved in calcium-entry mediated events (see for example, Trevillyanet al. (2001) J. Biol. Chem. 276:48118-26). The assays described hereincan be used with cells that have been treated with or contacted with atest agent, or that express an altered amount of a test molecule (suchas a molecule involved in calcium regulation, including a STIM orSTIM-like protein), or with control cells. The assays can also beconducted in cells that have been stimulated with a physiological ornon-physiological activator, or in unstimulated cells. The following arerepresentative assays for molecules involved in calcium-entry mediatedevents and are meant to be exemplary only. Other assays for thesemolecules and assays for other molecules involved in calcium-entrymediated events can also be employed in any of the screening and/ormodulation methods described herein.

β-hexosaminidase Release

In mast cells, Ca²⁺ influx results in degranulation and release ofinflammatory mediators such as heparin, histamine and enzymes such asβ-hexosaminidase. Detecting and/or measuring release of such moleculescan thus be used to monitor intracellular calcium. For example, mediafrom mast cells can be collected. A suitable substrate forβ-hexosaminidase (e.g. p-nitrophenyl-acetyl-glucosamide) can then beadded and the absorbance of the resulting mixture assessed to measurethe relative amount of β-hexosaminidase activity in the samples (see forexample, Example 10, and also, Funaba et al. (2003) Cell Biol.International 27:879-85).

Calcium/Calmodulin-Dependent CaN Phosphatase Activity

The phosphatase calcineurin (CaN) dephosphorylates various proteins,affecting their activity and localization. CaN activity can be assessedby incubating purified CaN and a CaN substrate, for example aradiolabeled peptide corresponding to a sequence in the R_(II) subunitof cAMP-dependent kinase, either with or without a test agent or testmolecule (see, Trevillyan et al. (2001) J. Biol. Chem 276:48118-26). Thelevel of radiolabeled peptide and/or the amount of free inorganicphosphate released can be measured to assess CaN dephosphorylationactivity.

NFAT Transcriptional Activity

The NFAT (nuclear factor of activated T cells) transcription factorregulates a number of genes in response to intracellular calcium levels.For example, NFAT proteins regulate the transcription of cytokine genesinvolved in the immune response. Promoters from NFAT-regulated genes,and/or regulatory regions and elements from these genes, can be used tomonitor NFAT regulated expression and thereby monitor intracellularcalcium. Reporter gene fusions can be constructed with NFAT regulatedpromoters or NFAT-regulated elements operably linked to a reporter genesuch as luciferase, β-galactosidase, green fluorescent protein (GFP) orany other known reporter in the art (see for example, Published U.S.Application no. 2002-0034728). The amount of reporter protein oractivity is a measure of NFAT activity.

NFAT Phosphorylation

NFAT activation is regulated primarily through its phosphorylation,which in turn regulates its subcellular localization. In unstimulatedcells, NFAT is a hyperphosphorylated cytosolic protein. An elevation inintracellular Ca²⁺, induced by a variety of mechanisms, increases theactivity of the Ca²⁺-calmodulin-dependent phosphatase, calcineurin.Activated calcineurin dephosphorylates multiple serine residues withinthe regulatory region of the NFAT molecule. NFAT is rephosphorylated inresponse to decreases in Ca²⁺ levels or CaN inhibition.

The phosphorylation state of NFAT can be monitored for example, byexpressing a detectably tagged NFAT protein in cells, such as a His₆tagged-NFAT. Tagged NFAT can be purified from cells using Ni²⁺chromatography and subjected to gel electrophoresis and staining orwestern blotting. More highly phosphorylated forms of NFAT can bedistinguished by their slower migration. The state of phosphorylatedNFAT can be used as a measure of NFAT activation (see, Trevillyan et al.(2001) J. Biol. Chem 276:48118-26).

NFAT Nuclear Localization

NFAT localization between the cytoplasm and nucleus is regulated by thephosphorylation state of NFAT. Phosphorylation of NFAT prevents nuclearlocalization by masking the nuclear localization sequence. NFAT nuclearlocalization can be monitored, for example, by expressing fluorescentlytagged NFAT, for example, GFP-NFAT, in cells. Confocal microscopy can beused to monitor nuclear localization of the tagged NFAT (see, Trevillyanet al. (2001) J. Biol. Chem 276:48118-26).

Cytokine Secretion

Cytokine secretion, such as IL-2 secretion, can be monitored usingprotein detection assays. For example, supernatant can be collected fromimmune cells. An ELISA assay or other suitable format with IL-2antibodies can be used to detect and/or measure the amount of IL-2secreted as compared to control cells (see EXAMPLE 9). Secretion ofother cytokines, for example, TNFα, can also be detected in similarassays.

Cytokine Expression

Expression of cytokines, such as IL-2, can be assessed either directlyor indirectly in cells. For example, in indirect methods, an IL-2promoter can be operably linked to a reporter gene such as luciferase orβ-galactosidase, and the reporter construct introduced into cells.Reporter gene expression can be monitored and compared to geneexpression in control cells (see, Trevillyan et al. (2001) J. Biol. Chem276:48118-26). Alternatively, expression of endogenous or recombinantIL-2 mRNA or protein can be assessed.

T Cell Proliferation

Cytokines such as IL-2 are necessary for T-cell proliferation inresponse to mitogen or alloantigen stimulation, and thus T-cellproliferation is altered by changes in cytokine expression or secretion.T cells can be induced, such as with concanavalin A or alloreactivelymphocytes and T cell proliferation measured, for example, bysubjecting cells to a pulse of ³H-thymidine and measuring ³H-thymidineincorporation (see, Trevillyan et al. (2001) J. Biol. Chem276:48118-26).

d. Systems

Also provided herein are systems for use in identifying an agent thatmodulates intracellular, including, for example, cytoplasmic, calcium.Such systems include a protein or portion thereof (and/or nucleic acidencoding a protein or portion thereof) involved in modulatingintracellular calcium that has an amino acid sequence homologous to anamino acid sequence of a mammalian, e.g., rodent or human, stromalinteracting molecule (STIM) protein and/or a protein encoded by aDrosophila gene that, when altered in its expression in a Drosophilacell, results in altered store-operated calcium entry into the cell,altered calcium levels in or movement of calcium into, out of or withinan intracellular calcium store and/or altered cytosolic calciumbuffering. One such Drosophila gene is CG9216. In a particularembodiment, the protein is one that is involved in modulatingintracellular calcium (and, in particular embodiments is involved in,participates in and/or provides for store-operated calcium entry,movement of calcium into, out of or within an intracellular organelle orcalcium store, modulation of intracellular store or organelle calciumlevel, and/or cytosolic calcium buffering) and that has an amino acidsequence that is at least about 20%, or at least about 25%, or at leastabout 30%, or least about 35%, or at least about 39%, or at least about40%, or at least about 45%, or at least about 47%, or at least about50%, or at least about 55%, or at least about 60%, or at least about65%, or at least about 70%, or at least about 75%, or at least about80%, or at least about 85%, or at least about 90%, or at least about 95%or more homologous to an amino acid sequence of the protein encoded bythe coding sequence of Drosophila gene CG9126 and/or a mammalian STIMprotein, e.g., human or rodent (such as rat) STIM1. The particularhomology can depend on the particular protein, e.g., species, that ishomologous to the specified proteins and the extent of the specifiedproteins to which the particular protein is homologous. In particularembodiments, the protein is at least 45% or more homologous to theprotein encoded by the coding sequence of Drosophila gene CG9126 and/ora mammalian STIM protein, e.g., human or rodent (such as rat) STIM1.Such proteins may be homologous to the specified proteins over at leastabout 25%, or at least about 30%, or at least about 35%, or at leastabout 40%, or at least about 45%, or at least about 50%, or at leastabout 52%, or least about 55%, or at least about 60%, or at least about65%, or at least about 70%, or at least about 75%, or at least about80%, or at least about 84%, or at least about 85%, or at least about90%, or at least about 95% or more of a specified protein. In particularembodiments, the protein is homologous to a specified protein over atleast about 52% or more of a specified protein.

In another particular embodiment of the above systems, a protein, orportion thereof (and/or nucleic acid encoding a protein or portionthereof), of the system is involved in intracellular calcium modulation(and in particular embodiments, is involved in, participates in and/orprovides for store-operated calcium entry, movement of calcium into, outof or within an intracellular organelle or calcium store, modulation ofintracellular store or organelle calcium level, and/or cytosolic calciumbuffering) that is at least about 45% homologous over at least about 52%of the protein encoded by the coding sequence of Drosophila gene CG9126and/or a mammalian STIM protein, e.g., human or rodent (such as rat)STIM1. Proteins homologous to the protein encoded by the coding sequenceof Drosophila gene CG9126 and/or a mammalian STIM protein, e.g., humanor rodent (such as rat) STIM1, include, but are not limited to, theproteins listed in Table 3. In other embodiments, other proteins thatmay be used in the systems provided herein include, but are not limitedto, proteins involved in intracellular calcium modulation that aresubstantially homologous to the proteins listed in Table 3.

In one embodiment of the systems for use in identifying an agent thatmodulates intracellular calcium, the system includes a cell, or portionthereof, containing one or more STIM proteins or STIM-like proteins, orportion thereof. Examples of STIM or STIM-like proteins include, but arenot limited to, the following: STIM proteins (e.g., of Drosophila (SEQID NOS: 2, 76, 78, 80 and 81) and C. elegans (SEQ ID NO: 14)); STIM1 orSTIM1-like proteins (e.g., of Homo sapiens (SEQ ID NOS: 4, 50, 83, 84),reference STIM1 (SEQ ID NO: 52) and Mus musculus (SEQ ID NOS:10, 56 and85)); STIM2 or STIM2-like proteins (e.g., of Homo sapiens (SEQ ID NOS:6, 62, 87 and 88), Mus musculus (SEQ ID NOS: 12 and 68), rat (SEQ ID NO:72) and partial protein sequences from hamster SEQ ID NO:96 and from rat(SEQ ID NO:98)).

In a particular embodiment of the system, the protein can be containedin a cell, or portion thereof (e.g., a membrane or intracellularorganelle). A cell of the system can be an isolated cell (or can be in acell culture) that endogenously expresses such protein(s) and/or canexpress such proteins from heterologous nucleic acid (e.g., recombinantexpression) as described above with respect to the methods foridentifying agents. In a particular embodiment, the cell is one thatoverexpresses the protein. Systems in which the cell recombinantlyexpresses the protein can be such that the cell is an isolated cell oris in a cell culture or are contained within an animal, in particular, anon-human animal, e.g., a non-human mammal.

The protein, or portion thereof (and/or nucleic acid encoding a proteinor portion thereof), and/or cell, or portion thereof, of particularembodiments of the systems can be contained in a medium that contains anagent that provides for passive or active intracellular calcium storereduction or depletion (e.g., thapsigargin or other non-physiologicalactivator of calcium store depletion) and/or that contains a molecule ormolecules that facilitate monitoring or measurement of intracellularcalcium and/or calcium movement. Such molecules include fluorescent (orotherwise labeled) calcium indicators, trivalent cations, divalentcations other than calcium and calcium-buffering agents, e.g., calciumchelators.

E. Methods of Modulating Intracellular Calcium

Provided herein are methods for modulating intracellular calcium.Modulation of intracellular calcium can be any alteration or adjustmentin intracellular calcium including but not limited to alteration ofcalcium concentration or level in the cytoplasm and/or intracellularcalcium storage organelles, e.g., endoplasmic reticulum, alteration inthe movement of calcium into, out of and within a cell or intracellularcalcium store or organelle, alteration in the location of calcium withina cell, and alteration of the kinetics, or other properties, of calciumfluxes into, out of and within cells. In particular embodiments,intracellular calcium modulation can involve alteration or adjustment ofstore-operated calcium entry, cytosolic calcium buffering, calciumlevels in or movement of calcium into, out of or within an intracellularcalcium store or organelle, and/or basal or resting cytosolic calciumlevels. In some embodiments, modulation of intracellular calcium caninvolve an alteration or adjustment in receptor-mediated ion (e.g.,calcium) movement, second messenger-operated ion (e.g., calcium)movement, calcium influx into or efflux out of a cell, and/or ion (e.g.,calcium) uptake into or release from intracellular compartments,including, for example, endosomes and lysosomes.

1. Modulating a Protein(s), and/or Nucleic Acid Encoding a Protein,Involved in Modulating Intracellular Calcium

Methods of modulating intracellular calcium include a step of modulatinga protein involved in modulating intracellular calcium. Thus, methods ofmodulating intracellular calcium provided herein can be methods ofspecifically or selectively modulating intracellular calcium, incontrast to modulation of intracellular calcium that may occur as one ofseveral effects that a general, non-specific manipulation of a cell mayelicit. Modulating a protein involved in modulating intracellularcalcium can include, for example, modulating the level, expression,functioning, molecular interactions and/or activity of a protein orportion thereof (and/or nucleic acid encoding one or more proteins orportion thereof) involved in modulating intracellular calcium. Theprotein involved in modulating intracellular calcium can be, forexample, an ion transport protein, a component of an ion transportprotein complex, a modulatory or regulatory protein, a receptor, acalcium-binding protein or a protein that regulates any such proteins.

In one embodiment, the method includes a step of specifically modulatinga particular protein involved in modulating intracellular calcium. Inspecifically modulating a particular protein involved in modulatingintracellular calcium, the particular protein is targeted formodulation. Specific modulation of a particular protein involvesselectively (and typically directly) modulating the protein and thusalso modulating any particular intracellular calcium modulation processor pathway in which the particular protein may be involved. Inembodiments of the methods for modulating intracellular calcium in whicha particular protein is specifically modulated, the modulation of theprotein can be such that the associated modulation of intracellularcalcium occurs through a process that is primarily initiated through adirect effect on the particular protein. Specific modulation of aparticular protein can be such that the particular protein, and thus aparticular pathway or cascade or intracellular calcium-modulatingprocess in which it is involved, is directly modulated, altered oraffected to a greater extent than other proteins that are not involvedin the particular intracellular calcium-modulating process or signalingpathway that the particular protein is involved in.

In one embodiment, the method may include a step of contacting orexposing a cell, or portion thereof (e.g., a membrane or intracellularcalcium store or organelle), with an agent that modulates the level,expression, functioning, molecular interactions and/or activity of oneor more proteins (or a gene or nucleic acid encoding one or moreproteins) involved in modulating intracellular calcium. In particularembodiments, the agent is one that specifically modulates a particularprotein involved in modulating intracellular calcium. The cell, orportion thereof, can be one that contains the protein involved inmodulating intracellular calcium. The cell can be one, for example, thatexhibits altered intracellular calcium. The cell can be an isolatedcell, a cell in culture, in a tissue or in an organism.

Particular proteins (and/or nucleic acids encoding proteins) involved inmodulating intracellular calcium (and, in particular, that are involvedin, participate in, and/or provide for store-operated calcium entry,movement of calcium into, out of or within an intracellular calciumstore or organelle, modulation of calcium levels in intracellularcalcium stores or organelles, and/or cytosolic calcium buffering) areprovided herein. The proteins provided herein and described above (andelsewhere herein) can be used in methods of modulating intracellularcalcium provided herein. Thus, for example, the protein can be one thatis homologous to an amino acid sequence of the protein encoded by thecoding sequence of Drosophila gene CG9126 and/or to a mammalian stromalinteracting molecule (STIM) protein, e.g., human or rodent (such as rat)STIM1. In particular embodiments, the protein is one that is involvedin, participates in and/or provides for store-operated calcium entry,movement of calcium into, out of or within an intracellular calciumstore or organelle, modulation of calcium levels in intracellularcalcium stores or organelles, and/or cytosolic calcium buffering. Inparticular embodiments, the protein is one of the proteins (or issubstantially homologous to one of the proteins) listed in Table 3. Theprotein can be, for example, a STIM or STIM-like protein, including aSTIM1, STIM2, DSTIM or CSTIM protein. In one embodiment of the methods,the protein is a STIM1 protein, for example, a mammalian STIM1 protein.

The step of modulating one or more proteins involved in modulatingintracellular calcium can be performed in a variety of ways and mayinvolve the use of a number of agents. Agents that can be used in themethods include agents described herein for modulating a proteininvolved in modulating intracellular calcium as well as agents that canbe identified using the methods provided herein for screening for oridentifying particular agents (e.g., agents that interact with ormodulate the level, activity and/or interactions of proteins providedherein) that modulate intracellular calcium. In particular embodiments,the agent is one that specifically modulates a particular proteininvolved in modulating intracellular calcium. For example, the agent canbe one that specifically or selectively binds to or interacts with aprotein involved in modulating intracellular calcium or a gene (orportion thereof) or nucleic acid encoding such a protein and/or that isbased on a specific feature, e.g., amino acid or nucleic acid sequence,of the protein. Included among such agents are compounds thatspecifically or selectively bind to or interact with such a protein,antibodies that specifically or selectively recognize such a protein,peptides that specifically alter (e.g., disrupt or inhibit) interactionof such a protein with another molecule, and nucleic acids thatspecifically bind to or hybridize with a gene, or portion thereof, ornucleic acid (e.g., DNA, RNA, mRNA) encoding such a protein.

In one example of modulating such proteins, the level and/or expressionof a protein (and/or nucleic acid encoding a protein) involved inmodulating intracellular calcium can be altered, e.g., in a cell orportion thereof. For example, the level and/or expression of a protein(and/or nucleic acid encoding a protein) involved in modulatingintracellular calcium can be increased or reduced. In a particularembodiment, the level and/or expression of a protein (and/or nucleicacid encoding a protein) involved in modulating intracellular calcium isspecifically or selectively altered.

In one method for altering the level of a protein in a cell or portionthereof, the level of expression of nucleic acid encoding the protein isaltered. The level of expression of nucleic acid encoding the proteincan be specifically or selectively altered. Agents for use in alteringnucleic acid expression include agents that functionally bind totranscription regulatory sequences that are operably linked to nucleicacid encoding a protein, and nucleic acids. Nucleic acid agents can alsobe used to alter protein levels. Such nucleic acid agents includeinhibitory nucleic acids that reduce the level of and/or reduce orprevent the translation of mRNA coding for a protein. Such agentsinclude RNA, e.g., antisense RNA and molecules that can be used in RNAinterference-based processes, such as double-stranded RNA (dsRNA) andsmall interfering RNAs. Expression of a nucleic acid encoding a proteincan also be reduced or eliminated by introducing a nucleic acid, such asa cDNA or expression vector, that serves to disrupt (e.g., throughhomologous recombination) the already present DNA encoding the proteinsuch that the level of the protein is decreased in a cell or the proteinthat is produced is altered (e.g., truncated) such that it is notfunctional or active with respect to its intracellularcalcium-modulating activity. The level of a protein in a cell can alsobe altered by altering, e.g., increasing, the number of copies ofnucleic acid encoding the protein present in the cell. Thus, forexample, an agent can be a nucleic acid, e.g., genomic DNA, cDNA orexpression vector, that is introduced into the cell for expression ofthe protein therein, thereby increasing the level of the protein in thecell.

Agents that can affect expression of a nucleic acid encoding particularproteins provided herein are described herein and/or can be identifiedand produced using methods described herein and/or known in the art. Forexample, an agent that modulates transcription regulatory elements of aSTIM1 gene can be identified as an agent that alters the level ofexpression of a reporter molecule operably linked to STIM1 gene promotersequences. In addition, numerous examples of nucleic acids encoding STIMand STIM-like proteins (and portions thereof) are provided herein andcan be used in designing and producing cDNAs, expression vectors,antisense RNAs and dsRNAs using methods known in the art.

In another example of modulating a protein involved in modulatingintracellular calcium, an activity of a protein (and/or nucleic acidencoding a protein) involved in modulating intracellular calcium can bealtered, e.g., in a cell or portion thereof, by methods other thanthrough altering protein (and/or nucleic acid) levels. For example, anactivity of a protein (and/or nucleic acid encoding a protein) involvedin modulating intracellular calcium can be increased or reduced. Anactivity of a protein (and/or nucleic acid encoding a protein) involvedin modulating intracellular calcium can be specifically or selectivelyaltered. For example, the activity of a particular protein (and/ornucleic acid encoding a particular protein) can be altered to a greaterextent than the activity of other proteins such as proteins that are notinvolved in a particular intracellular calcium-modulating process orpathway that the particular protein is involved in. The activity can beone that is involved in modulating intracellular calcium either directly(e.g., has an immediate direct effect) or indirectly (e.g., an upstreamevent in a pathway that ultimately affects intracellular calcium).Alterations in an activity of the protein include, but are not limitedto, increases and decreases in an activity and/or functioning of theprotein. Activities of calcium-modulating proteins include, but are notlimited to, calcium transport, calcium binding, other interactions(e.g., protein-protein interactions) and regulation ofcalcium-modulating proteins. Thus, agents that modulate theseactivities, or any other activity involved in intracellular calciummodulation, can be used in the methods provided herein. For example,antibodies or other proteins that specifically bind to the protein andmodulate such activities can be agents used in the methods. Examples ofparticular antibodies that recognize and bind to STIM proteins aredescribed herein. An antibody or other protein may, for instance, bindto a site of a regulatory protein and reduce or eliminate its binding tothe protein it regulates, thereby reducing its regulatory activity. Apeptide based on a particular amino acid sequence of a protein (e.g.,the sequence of a protein-protein interaction domain) can be an agentthat may, for example, alter (e.g., disrupt or inhibit) interaction of aprotein with a binding or interacting partner. Examples of a number ofdomains, including protein-protein interaction domains, of STIM proteinsare described herein and with reference to particular STIM amino acidsequences. Molecules, such as small organic molecules, can also beagents that may, for example, reduce or increase calcium transport by anion transport protein and thus can be agents used in the methods. Agentsthat modulate particular proteins provided herein can also be identifiedusing methods described herein and/or known in the art. Such methodsinclude binding and interaction assays and assays for detectingdisruption or inhibition of binding (e.g., STIM1 homotypic andSTIM1/STIM2 hetero-oligomeric interactions) described above.

2. Altered Intracellular Calcium

In one embodiment of the methods of modulating intracellular calcium,one or more proteins (and/or nucleic acid encoding one or more proteins)involved in modulating intracellular calcium is (are) modulated in aparticular cell (or portion thereof): a cell (or portion thereof) thatexhibits altered intracellular calcium. Altered intracellular calcium ina cell can be differences in intracellular calcium relative to a cellrecognized as normal with respect to intracellular calcium, e.g. a cellthat exhibits overall calcium homeostasis and controlled, regulatedcalcium responses to activation of processes that involve calciummobilization. Thus, altered intracellular calcium can be aberrant,abnormal, or defective intracellular calcium or intracellular calciumregulation. This embodiment of the methods can involve providing orselecting a cell that exhibits altered intracellular calcium. A varietyof methods are described herein and/or known in the art for assessingintracellular calcium, and numerous aspects of intracellular calciumregulation or modulation, in a cell or portion thereof to determine if acell exhibits altered intracellular calcium.

Altered intracellular calcium at a basic level can be an alteration incalcium levels and/or calcium location in a cell and/or movement ofcalcium into, out of or within a cell or intracellular organelle. Thus,in one example, an alteration in intracellular calcium can be analteration in the calcium level within an intracellular organelle orcalcium storage compartment or an alteration in basal or restingcytosolic calcium levels. Altered intracellular calcium can occurthrough an alteration in any one or more aspects of intracellularcalcium modulation, including alterations in store-operated calciumentry, calcium buffering, receptor-mediated and secondmessenger-operated ion (e.g., calcium) movement. For example, analteration of store-operated ion flux into a cell can be a complete ornearly complete elimination of the activity, a reduction of theactivity, an alteration in properties or characteristics (e.g., currentproperties or sensitivities) of the activity or an increase in theactivity relative to the activity in a cell that does not have alteredstore-operated calcium entry activity.

Cells that have altered intracellular calcium can have phenotypesindicative of the alteration(s). For example, T cells, fibroblasts, andin some cases B cells, from patients with a primary T cellimmunodeficiency or severe-combined immunodeficiency (SCID) having aprincipal defect in T cell activation show a strong defect instore-operated calcium entry (Feske et al. (2001) Nature Immunol.2:316-324; Paratiseti et al. (1994) J. Biol. Chem. 269:32327-32335; andLe Deist et al. (1995) Blood 85:1053-1062). In another example,psoriatic keratinocytes (from patients with plague-type psoriasis) havedisturbed (down-regulated) store-operated calcium entry and slightlylower calcium levels in calcium stores relative to normal keratinocytes(Karvonen et al. (2000) J. Invest. Dermatol. 114:693-700). In a furtherexample, B cells from a patient with Scott syndrome (an inheriteddisorder of the migration of phosphatidylserine toward the exoplasmicleaflet of the plasma membrane of stimulated white blood cells) show analteration (reduction) in store-operated calcium entry (Martin et al.(2000) Biochem. Biophys. Res. Commun. 279:639-645; Martinez et al.(1999) Biochemistry 38:10092-10098).

Provided herein are methods of modulating intracellular calcium in aparticular selected cell which is one that has altered intracellularcalcium. The methods include a step of modulating one or more proteins(and/or a gene or nucleic acid encoding one or more proteins) involvedin modulating intracellular calcium, and, in particular, proteinsprovided herein, including STIM and STIM-like proteins. In particularembodiments, the selected cell is one that exhibits alteredstore-operated calcium entry, calcium buffering, and/or calcium levelsin or movement of calcium into, out of or within an intracellularcalcium store or organelle. In particular embodiments, the cell is animmune system cell (e.g., a lymphocyte, white blood cell, T cell, Bcell), a fibroblast (or a cell derived from a fibroblast), or anepidermal, dermal or skin cell (e.g., a keratinocyte). The step ofmodulating one or more proteins (or nucleic acid encoding one or moreproteins) involved in modulating intracellular calcium can involve, forexample, increasing or reducing the level, expression of, an activityof, function of and/or molecular interactions of a protein. Forinstance, if a cell exhibits a deficit in some aspect of intracellularcalcium, e.g., store-operated calcium entry, then modulating may involveincreasing the level of, expression of, an activity or function of or amolecular interaction of a protein (and/or nucleic acid encoding aprotein). If a cell exhibits an increase in calcium levels or lack ofregulation of an aspect of intracellular calcium modulation, thenmodulating may involve reducing the level of, expression of, an activityor function of or a molecular interaction of a protein (and/or nucleicacid encoding a protein).

F. Methods of Screening for or Identifying Agents for the Treatment of aDisease or Disorder

Provided herein are methods for screening for or identifying agents orcandidate agents for the treatment or prevention of diseases ordisorders. Many of these methods are also methods for screening for oridentifying an agent that modulates intracellular calcium. In a firstembodiment, the methods provided herein and described above (andelsewhere herein) for screening for or identifying an agent thatmodulates intracellular calcium can be used to identify agents andcandidate agents for the treatment of diseases and disorders involvingor characterized at least in part by calcium dyshomeostasis ordysregulation or alterations in intracellular calcium, including, forexample, alterations in calcium signaling. Therapeutic potential ofcandidate agents can be further evaluated in disease models, whichinclude cell- and organism-based systems. Models for diseases anddisorders involving alteration of intracellular calcium are describedherein and/or known in the art.

Methods provided herein of identifying or screening for agents thatmodulate intracellular calcium, and methods provided herein ofidentifying or screening for agents or candidate agents for thetreatment or prevention of diseases/disorders also include methods whichutilize particular cells and/or organisms, i.e., cells and/or organismsthat have altered intracellular calcium (or that exhibit calciumdysregulation) and/or that are models of diseases or disorders that (1)involve or are characterized at least in part by calcium dyshomeostasisor dysregulation or alterations in intracellular calcium or (2) involvean alteration or aberrant functioning of a cellular process which relieson or is regulated by intracellular calcium. Such cells or organisms areknown in the art and/or described herein. In one embodiment of suchmethods, the effect of a test agent on intracellular calcium is directlyor indirectly monitored or assessed using a cell, or portion thereof,containing a polymorphic form of one or more proteins (or portionthereof) involved in modulating intracellular calcium (and/or apolymorphic form of a nucleic acid, such as a gene, cDNA or RNA, orportion(s) thereof, encoding such protein) that is not a wild-type formof the protein (or nucleic acid) and that is associated with analteration in intracellular calcium in the cell, or portion thereof. Thealteration in intracellular calcium can be relative to intracellularcalcium in a cell, or portion thereof, that contains a wild-type orreference form of the one or more proteins (or nucleic acid, or portionthereof, encoding a protein) involved in modulating intracellularcalcium. In particular embodiments, the alteration in intracellularcalcium can be an alteration of store-operated calcium entry, cytosoliccalcium buffering, calcium levels in or movement of calcium into, out ofor within an intracellular calcium store or organelle (e.g., endoplasmicreticulum) and/or maintenance of resting cytosolic calcium levels. In aparticular embodiment, the effect(s) of a test agent on store-operatedcalcium entry, cytosolic calcium buffering, calcium levels in ormovement of calcium into, out of or within an intracellular calciumstore or organelle (e.g., endoplasmic reticulum) and/or restingcytosolic calcium levels is (are) assessed. In this embodiment, the testagent can be any agent. The cell, or portion thereof, used in the methodcan be an isolated cell, one or more cells in a culture or collection ofcells or can be in a tissue or organism (e.g., an animal model).

In another embodiment of the methods for screening for or identifying anagent or candidate agent for the treatment of a disease or disorder, themethod can be conducted using a particular test agent: one that bindsto, interacts with and/or modulates interactions, activities, levels orany physical, structural or other property of a protein involved inmodulating intracellular calcium. In this embodiment, the methodincludes monitoring or assessing the effects of such a test agent onintracellular calcium and/or a disease/disorder phenotype of a cell- ororganism-based model of a disease or disorder that (1) involves or ischaracterized at least in part by calcium dyshomeostasis ordysregulation or alterations in intracellular calcium (including, forexample, alterations in calcium signaling) or (2) involves an alterationor aberrant functioning of a cellular process which relies on or isregulated by intracellular calcium. In particular embodiments, theeffect(s) of a test agent on store-operated calcium entry, cytosoliccalcium buffering, calcium levels in or movement of calcium into, out ofor within an intracellular calcium store or organelle (e.g., endoplasmicreticulum) and/or resting cytosolic calcium levels is (are) assessed.Test agents used in this embodiment of the methods can be agents thatare already known to bind to, interact with and/or modulate a particularprotein involved in modulating intracellular calcium or can beidentified as such using methods described herein and/or known in theart. Methods for identifying such test agents include methods describedherein above, such as, for example, binding, interaction, level andactivity assays (and, in particular, STIM protein binding andinteraction assays, activity assays and assays for detecting disruptionor inhibition of binding, e.g., STIM1 homotypic and STIM1/STIM2hetero-oligomeric interactions, described above.) The method canoptionally include a step of identifying an agent that can bind to,interact with and/or modulate interactions, activities, levels or anyphysical, structural or other property involved in modulatingintracellular calcium. This optional step can be performed prior to orconcurrently with the step of monitoring the effects of the test agenton intracellular calcium and/or a disease/disorder phenotype.

In particular embodiments of any of the methods of screening for oridentifying an agent or candidate agent for the treatment of a diseaseor disorder provided herein, the protein(s) involved in modulatingintracellular calcium can be a protein that is involved in, participatesin and/or provides for store-operated calcium entry, cytosolic calciumbuffering, maintenance of resting cytosolic calcium levels and/ormodulation of calcium levels in, or movement of cations into, out of orwithin an intracellular organelle or calcium store, such as, forexample, the endoplasmic reticulum. In particular embodiments, theprotein involved in modulating intracellular calcium is an ion transportprotein, such as, for example, an ion transport protein that is involvedin, participates in and/or provides for store-operated calcium entry ormovement of calcium into, out of or within the endoplasmic reticulum orother calcium store. In one embodiment, the protein involved inmodulating intracellular calcium is a component of a store-operatedcalcium entry channel complex (e.g., a multimeric complex containingmultiple subunits of the same and/or different proteins). In anotherembodiment the protein involved in modulating intracellular calcium is amodulatory protein. A protein involved in modulating intracellularcalcium (and/or nucleic acid, or portion(s) thereof, encoding a proteininvolved in modulating intracellular calcium) may be contained in a cellor portion thereof, such as, for example, a cell membrane (e.g., plasmamembrane or an intracellular membrane). The methods may be performed inparticular embodiments under conditions that permit specific evaluationof store-operated ion flux or movement, resting cytosolic calciumlevels, cytoplasmic calcium buffering and/or cation levels in, ormovement into, out of or within an intracellular organelle or calciumstore, such as, for example, the endoplasmic reticulum.

A protein involved in modulating intracellular calcium that is used inthe methods (or on which a method is based) can be a full-length orcomplete protein (e.g., a protein that contains the complete amino acidsequence encoded by a gene, cDNA or RNA or a complete amino acidsequence that lacks sequences that are removed during processing of theprotein, including removal of a signal sequence, such as may occur intransport of a protein to a particular cellular location, or processingto remove a pre- and/or pro-sequence of a protein) or a portion of acomplete protein. In embodiments of the methods that involve assessing afunctional activity of the protein, the protein used in the method canbe a portion of a full-length protein that is associated with, orexhibits or is sufficient for producing the functional activity, e.g.,an intracellular calcium-modulating activity. In embodiments of themethods that involve assessing a property of the protein that is notnecessarily the intracellular calcium-modulating activity of theprotein, the protein used in the method can be a portion of afull-length protein that is associated with the particular propertybeing assessed, e.g., binding properties, and can be, for example, aparticular domain of the protein. Similarly, when a nucleic acid, orportion(s) thereof, encoding a protein involved in modulatingintracellular calcium is used in the methods, the nucleic acid can be acomplete gene, e.g., including transcriptional regulatory sequences, acomplete protein coding sequence, e.g., cDNA or RNA, or portion(s) ofthese (e.g., a portion encoding a functional domain of a protein).

In particular embodiments of the methods for screening for oridentifying an agent or candidate agent for the treatment of a diseaseor disorder, a protein involved in modulating intracellular calcium canbe a protein (or portion thereof) involved in modulating intracellularcalcium as provided herein and described above (and elsewhere herein).Thus, for example, the protein can be one that is homologous to an aminoacid sequence of the protein encoded by the coding sequence ofDrosophila gene CG9126 and/or to a mammalian stromal interactingmolecule (STIM) protein, e.g., human or rodent (such as rat) STIM1. Inparticular embodiments, the protein is one that is involved in,participates in and/or provides for store-operated calcium entry,movement of calcium into, out of or within an intracellular calciumstore or organelle, modulation of calcium levels in intracellularcalcium stores or organelles, and/or cytosolic calcium buffering. Inparticular embodiments, the protein is one of the proteins (or issubstantially homologous to one of the proteins) listed in Table 3. Theprotein can be, for example, a STIM or STIM-like protein, including aSTIM1, STIM2, DSTIM or CSTIM protein. In one embodiment of the methods,the protein is a STIM1 protein, for example, a mammalian STIM1 protein.

1. Cells and Cell Models

Cell that can be used in embodiments of the methods for screening for oridentifying agents or candidate agents for the treatment of diseases anddisorders include cells that exhibit an alteration in intracellularcalcium or that exhibit calcium dysregulation or dyshomeostasis. Suchcells can also be used in elucidating the mechanisms underlying calciumdyshomeostasis or altered calcium signaling in a cell as well as indissecting processes involved in intracellular calcium regulation. Cellsfor use in the methods also include cell models of diseases or disordersinvolving or characterized at least in part by calcium dyshomeostasis ordysregulation or alterations in intracellular calcium, including, forexample, alterations in calcium signaling.

Cells that exhibit an alteration in intracellular calcium or thatexhibit calcium dysregulation or dyshomeostasis can include cells thatcontain a polymorphic form of one or more proteins (or portion thereof)involved in modulating intracellular calcium (and/or a polymorphic formof a nucleic acid, such as a gene, cDNA or RNA, or portion(s) thereof,encoding such protein) that is not a wild-type or reference form of theprotein (or nucleic acid). A polymorphic form of a protein or nucleicacid is a form that varies in amino acid or nucleotide sequence relativeto a reference or wild-type form. With respect to cells that exhibitaltered intracellular calcium and that contain a polymorphic form of aprotein and/or nucleic acid, a wild-type or reference form of theprotein or nucleic acid can be one that occurs in a cell that does notexhibit altered intracellular calcium or calcium dyshomeostasis. Awild-type form of a nucleic acid or protein can often be a predominantform in a population. A polymorphic or mutant protein can be one thathas an altered activity or function or one that has no activity or isnon-functional, particularly relative to a wild-type, reference orpredominant form of the protein. Cells that contain a polymorphic formof one or more proteins (or portion thereof) involved in modulatingintracellular calcium (and/or a polymorphic form of a nucleic acid, suchas a gene, cDNA or RNA, or portion(s) thereof, encoding such protein)that is not a wild-type form of the protein (or nucleic acid) can beevaluated using methods described herein and/or known in the art toidentify cells that exhibit an alteration in intracellular calcium inthe cell (or portion thereof). For example, an alteration inintracellular calcium can be identified through comparison ofintracellular calcium of the cell to intracellular calcium of a cell, orportion thereof, that contains a wild-type or reference form of the oneor more proteins (or nucleic acid, or portion thereof, encoding aprotein) involved in modulating intracellular calcium. Wild-type orreference proteins (and nucleic acids encoding such proteins) includeproteins (and nucleic acids) described herein and listed in Table 3. Inparticular embodiments, the alteration in intracellular calcium can bean alteration of store-operated calcium entry, cytosolic calciumbuffering, calcium levels in or movement of calcium into, out of orwithin an intracellular calcium store or organelle (e.g., endoplasmicreticulum) and/or maintenance of resting cytosolic calcium levels.

A polymorphic form of a sequence of nucleotides or amino acids can beany difference in the sequence relative to a reference or wild-typesequence. For example, a difference in sequence can be a singlenucleotide change (e.g., a SNP), a single amino acid change, a deletionor insertion of a single nucleotide or amino acid, or can be analteration of two or more consecutive nucleotides or amino acids (e.g.,insertions and deletions of a sequence of two or more nucleotides oramino acids). A polymorphic form of a protein can also be one which isshorter (or truncated) relative to a reference or wild-type amino acidsequence. A polymorphic form of a gene that encodes a particular proteincan be one that differs from a reference or wild-type form in anon-coding portion of the gene sequence (e.g., transcriptionalregulatory regions, such as promoters or enhancers, introns, anduntranslated sequences). Polymorphisms located in the primary RNAtranscript-encoding portion of a gene may affect the processing of thetranscript into mature RNA. For example, single nucleotide changes candisrupt the function of splicing enhancers located within codingsequences. Enhancers can be disrupted by single nonsense, missense ortranslationally silent point mutations. Single nucleotide polymorphismsthat function as exon splicing silencers have also been described whichcould alter splicing patterns of primary RNA transcripts.

Polymorphisms or mutations of particular proteins involved in modulatingintracellular calcium provided herein (or polymorphic genes or nucleicacids encoding such proteins or portions thereof) are known (see, e.g.,NCBI database of single nucleotide polymorphisms (SNPs) accessible atwww.ncbi.nlm.nih.gov/SNP/index.html). Cells containing a particularpolymorphic form of a protein (and/or gene or nucleic acid encoding aprotein) provided herein (e.g., a STIM or STIM-like protein, includingproteins listed in Table 3) can be cells that endogenously contain suchproteins (and/or nucleic acids) or that are generated throughrecombinant processes, such as, for example, through transfer of apolymorphic nucleic acid (or nucleic acid encoding a polymorphic proteinor portion thereof) into a host cell or through disruption of anendogenous gene encoding a wild-type form of a protein. Thus, cellmodels include recombinant cells that contain heterologous nucleic acidencoding a polymorphic form of one or more proteins involved inintracellular calcium modulation.

Cell models that exhibit an alteration in intracellular calcium or thatexhibit calcium dysregulation or dyshomeostasis can also includerecombinant cells in which expression of a protein involved inintracellular calcium modulation (which is either endogenously expressedin the cell or is expressed in the cell from a heterologous nucleicacid) has been altered or eliminated, such as by replacing or modifyingthe promoter region or other regulatory region driving expression ofnucleic acid encoding the protein. Such a cell can be produced, usingmethods known in the art and/or described herein, by introduction into acell of heterologous nucleic acid that either targets and alters DNAregulatory sequences associated with an endogenous gene or that linksDNA encoding the protein to a particular expression regulationsequence(s). Cell models including a recombinant cell in whichexpression of an endogenous protein involved in intracellular calciummodulation as identified above has been reduced or eliminated can beproduced by disruption or elimination (e.g., through gene knock-out,antisense RNA or RNA interference methods) of the gene or RNA encodingthe protein in the cell.

Any cell may be used in generating a cell models. In particularembodiments of the cell models, the cell is a neuronal, nervous system-or tissue-derived cell or a brain cell. For example, cells from a knowncell line can be used, such as from neuroblastoma SH-SY5Y cells,pheochromocytoma PC12 cells, neuroblastoma SK-N-BE(2)C cells, humanSK-N-MC neuroblastoma cells, SMS-KCNR cells, human LAN-5 neuroblastomacells, human GI-CA-N neuroblastoma cells, human GOTO neuroblastomacells, mouse Neuro 2a (N2A) neuroblastoma cells and/or human IMR 32neuroblastoma cells. Cell lines include HEK 293, CHO (including CHO-K1),LTK-, N2A, H6, HGB, and Drosophila S2 cells. In another embodiment, thecell can be an immune cell (e.g., lymphocyte, white blood cell, T cellor B cell), a fibroblast (or a cell derived from a fibroblast), anepidermal, dermal or skin cell (e.g. a keratinocyte), a blood cell, akidney or renal cell (e.g., mesangial cell), a muscle cell (e.g., asmooth muscle cell such as an airway (tracheal or bronchial) smoothmuscle cell) or an exocrine or secretory (e.g., salivary, includingparotid acinar and submandibular gland) cell. The generation,maintenance and use of such cell lines is well known.

Other cell models of particular diseases/disorders for use in themethods include (1) cells (e.g., T cells, fibroblasts, and B cells) fromsubjects with a primary T cell immunodeficiency or severe-combinedimmunodeficiency (SCID) having a principal defect in T cell activationshow a strong defect in store-operated calcium entry (Feske et al.(2001) Nature Immunol. 2:316-324; Partiseti et al. (1994) J. Biol. Chem.269:32327-32335; and Le Deist et al. (1995) Blood 85:1053-1062); (2)cells (e.g., psoriatic keratinocytes) from subjects with psoriasis(e.g., plague-type psoriasis) which have disturbed store-operatedcalcium entry and/or slightly lower calcium levels in calcium storesrelative to normal keratinocytes (Karvonen et al. (2000) J. Invest.Dermatol. 114:693-700); (3) cells (e.g., B cells) from subjects withScott syndrome (an inherited disorder of the migration ofphosphatidylserine toward the exoplasmic leaflet of the plasma membraneof stimulated white blood cells) which show an alteration (reduction) instore-operated calcium entry (Martin et al. (2000) Biochem. Biophys.Res. Commun. 279:639-645; Martinez et al. (1999) Biochemistry38:10092-10098); and (4) T cell (e.g., Jurkat T cell) mutants withdefective store-operated calcium entry (Fanger et al. (1995) J. CellBiol. 131:655-657). Cell models also include cells derived from animalmodels of particular diseases/disorders, including, for example, thosedescribed herein below (and elsewhere herein).

2. Animal Models

Animal models that can be used in embodiments of the methods forscreening for or identifying agents or candidate agents for thetreatment of diseases and disorders include animals (particularlynon-human animals) that have an alteration in intracellular calcium orthat have calcium dysregulation or dyshomeostasis. Animal models for usein the methods also include any animal model of a disease or disorderinvolving or characterized at least in part by calcium dyshomeostasis ordysregulation or alterations in intracellular calcium, including, forexample, alterations in calcium signaling.

Animal models that can be used in embodiments of the methods furtherinclude animals (particularly non-human animals) that have, in at leastsome of their cells, an alteration or defect in, or aberrant functioningof, a cellular process which relies on or is regulated by intracellularcalcium. Such cells need not have altered intracellular calcium orcalcium dyshomeostasis; yet, because intracellular calcium (andregulation thereof) plays an important role in such cellular processes,modulating intracellular calcium of such cells can be an effectivetreatment by compensating for the defect, counteracting the defect,reversing the defect or alleviating or eliminating the defect. Cellularprocesses that rely on or are regulated by intracellular calciuminclude, for example, cellular activation, gene expression, cellulartrafficking, and apoptosis. Diseases/disorders that involve defects thatmay be at least partially compensated for by modulation of intracellularcalcium include, but are not limited to: Autoimmune disorders, includingSjogren's syndrome (cytokines associated with lymphocyte invasion ofsalivary epithelial cells can reduce calcium mobilization in parotidcells; also, T-cell activation, including activation of transcriptionfactors, cytokine gene expression and cell proliferation, depends onsustained elevation of intracellular calcium level provided bystore-operated calcium influx), asthma (store-operated calcium entry mayplay an important role in mediating bronchial chonstriction andbronchial smooth muscle cell proliferation), glomerulonephritis andglomerular inflammation (changes in intracellular calcium, such as bystore-operated calcium entry, signal monocyte adhesion in a co-culturemodel of glomerular inflammation).

The animal models can also be used in elucidating the mechanismsunderlying calcium dyshomeostasis or altered calcium signaling in animalcells as well as in dissecting processes involved in intracellularcalcium regulation. Types of animal models include, but are not limitedto, non-human animals, such as non-human invertebrates and vertebratesand non-human mammals, rodents (e.g., mice, rat and hamster), cows,chickens, pigs, goats, dogs, sheep, insects, Drosophila, nematodes,worms, C. elegans, monkeys, gorillas, and other primates.

Animal models that exhibit an alteration in intracellular calcium orthat exhibit calcium dysregulation or dyshomeostasis can include animalsthat contain a polymorphic form one or more proteins (or portionthereof) involved in modulating intracellular calcium (and/or apolymorphic form of a nucleic acid, such as a gene, cDNA or RNA, orportion(s) thereof, encoding such protein) that is not a wild-type orreference form of the protein (or nucleic acid). Such a polymorphic ormutant protein can be one that has an altered activity or function orone that has no activity or is non-functional, particularly relative toa wild-type, reference or predominant form of the protein. Such animalscan be generated, for example, using methods known in the art forproducing transgenic animals that express an introduced transgene. Forexample, such animals can be prepared by “knock-in” methods in which theendogenous form (e.g., “normal”) of a gene encoding the protein(s) isreplaced by a variant, such a mutant, or other form. It is also possibleto replace one species', such as a rodent's, endogenous gene with a genefrom another species, such as from a human. Transgenic animals also canbe produced by non-homologous recombination into other sites in achromosome; including animals that have a plurality of integrationevents.

In another example of animal models that can be used in the methods, theexpression of nucleic acid encoding one or more proteins involved inmodulating intracellular calcium (e.g., particular proteins identifiedherein above) is altered or eliminated in at least some cells in theanimal. Alteration or elimination of expression of the protein(s) can beachieved in a number of ways. For example, expression can be altered byreplacing or modifying the promoter region or other regulatory region ofan endogenous gene encoding the protein(s) in the animal. Such an animalcan be produced using methods known in the art, e.g., by promotingrecombination between endogenous nucleic acid and an exogenous nucleicacid.

Increased expression of one or more of the proteins in a transgenicanimal can be achieved by altering or replacing an endogenous promoteror by introducing additional copies of nucleic acid encoding theprotein(s) into the animal. Reduction or elimination of expression ofone or more of the proteins can be achieved by disruption or “knockout”of endogenous genes in the animal. For example, such an animal caninitially be produced by promoting homologous recombination between agene of interest in its chromosome and the corresponding exogenous geneof interest that has been rendered biologically inactive (typically byinsertion of a heterologous sequence, e.g., an antibiotic resistancegene). In many organisms, e.g. C. elegans, it is possible to reduce oreliminate expression of a gene encoding a protein by introduction ofdouble-stranded RNA that contains sequence identical or complementary toat least a portion of the sequence of the gene of interest into theanimal.

Introduction of nucleic acids into cells for generation of transgenicanimals can be conducted using any known method of nucleic aciddelivery, including, but not limited to, microinjection, lipofection andother modes of nucleic acid delivery. The nucleic acids can beintroduced into cells such as, for example, germline cells or somaticcells, such as an embryonic stem cell. For example, the nucleic acid canbe introduced into a cell, such as an embryonic stem cell (ES), followedby injecting the ES cells into a blastocyst, and implanting theblastocyst into a foster mother, which is followed by the birth of atransgenic animal. Nuclear transfer methods can also be used to generatetransgenic animals. In these methods, nucleic acid being used togenerate a transgenic animal is introduced into a nuclear donor cellcontaining a totipotent nucleus, followed by transfer of the donornucleus into a recipient cell, e.g., an enucleated oocyte, which can betransferred to a recipient female for development into a transgenicanimal.

In one method of generating a “knock-out” transgenic animal, homologousrecombination is performed by transforming embryo-derived stem (ES)cells with a vector containing the insertionally inactivated gene ofinterest, such that homologous recombination occurs, followed byinjecting the ES cells into a blastocyst, and implanting the blastocystinto a foster mother, followed by the birth of the chimeric animal(“knockout animal”) in which a gene of interest has been inactivated(see Capecchi, Science 244: 1288-1292 (1989)). The chimeric animal canbe bred to produce homozygous knockout animals, which can then be usedto produce additional knockout animals. Knockout animals include, butare not limited to, mice, hamsters, sheep, pigs, cattle, and othernon-human mammals. The resulting animals can serve as models of diseasesinvolving altered expression of a protein involved in intracellularcalcium modulation. Such knockout animals can be used to screen foragents or candidate therapeutic agents or to test molecules that havealready been identified as candidate therapeutic agents for the abilityto treat or prevent such diseases or disorders.

Animal models also include non-transgenic animals. One example of suchan animal model that can be used in particular embodiments of themethods is SCID-human skin chimera mouse model of psoriasis andcutaneous inflammation (see, e.g., Raychaudhuri et al. (2001) British J.Dermatol. 144:931-939 and Nickoloff et al. (1995) Am. J. Pathol.146:580-588). This xenograft model can be generated by transplantationof psoriasis plaques onto a severe combined immunodeficient (SCID)mouse. Test agents can be delivered at the site of the lesion, and atherapeutic response can be determined, for example, through examinationand comparison of clinical, morphological and histological features ofthe lesions of animals that are treated and not treated with the testagent. Another example of an animal model that can be used in particularembodiments of the methods is a rodent model of airwayhyperresponsiveness (AHR), a characteristic of asthma. This model can begenerated, for example, by sensitization through immunization withovalbumin followed by exposure to aerosolized ovalbumin and challenge bycholinergic stimulation (e.g., via administration of methacholine oracetylcholine) (see, e.g., Xu et al. (2002) J. Appl. Physiol.93:1833-1840; Humbles et al (2002) Proc. Natl. Acad. Sci. 99:1479-1484).Airway hyperresponsiveness (which can be evaluated using methods knownin the art, e.g., using barometric plethysmography to record respiratorypressure curves and through measurement of pulmonary parameters such aspulmonary conductance and pulmonary compliance) can be assessed andcompared in animals treated and not treated with test agent. A furtherexample of an animal model that can be used in particular embodiments ofthe methods is a rodent model of mesangial proliferativeglomerulonephritis, which can be generated, for example, byadministration of anti-Thy1.1 antibody (see, e.g., Jefferson and Johnson(1999) J. Nephrol. 12:297-307). Any number of parameters indicative ofglomerulonephritis or renal dysfunction (e.g., mesangial cellproliferation, blood pressure, urinary protein excretion, creatinineclearance, glomerulosclerosis index and other parameters) can beevaluated and compared in animals treated with and not treated with testagent. The non-obese diabetic (NOD) mouse, an inbred mouse strain thatspontaneously develops an autoimmune diabetes that shares manyimmunogenetic features with Type 1 diabetes mellitus, is another exampleof an animal model that can be used in a particular embodiment of themethods. These mice also manifest many characteristics of autoimmuneexocrinopathy (such as Sjorgen's syndrome) including declining exocrinetissue secretory function (see, e.g., Humphreys-Beher and Peck (1999)Arch. Oral Biol. 44 Suppl 1:S21-25 and Brayer et al. (2000) J Rheumatol.27:1896-1904). Characteristics relevant to Sjorgen's syndrome (e.g.,lymphocytic infiltrates in exocrine glands (e.g., salivary and lacrimalglands), presence of dendritic cells and macrophages in submandibularglands, integrity of the lacrimal gland by measurement of basal andstimulated tear secretion, saliva flow rates and amylase activity) canbe evaluated and compared in animals treated with and not treated withtest agent. An animal (e.g., rodent) model of autoimmune disease canalso be used in particular embodiments of the methods. Such animalsinclude rat models available through the National Institutes of Health(NIH) Autoimmune Rat Model Repository and Development Center (Bethesda,Md.; accessible at www.ors.od.nih.gov/dirs/vrp/ratcenter). One rat modelof rheumatoid arthritis (RA) and related chronic/inflammatory autoimmunediseases is the collagen-induced arthritis (CIA) model (see, e.g.,Griffiths and Remmers (2001) Immunol. Rev. 184:172-183). Characteristicphenotypes of autoimmune disease (e.g. altered levels of immunereactivity to self-antigens, chronic inflammation ofautoantigen-expressing target organs, and activation and participationof invading mononuclear cells and tissue fibroblasts in organ damage)can be evaluated and compared in animals treated with and not treatedwith test agent. An animal (e.g., rodent) model of neuropathic orinflammatory pain can also be used in a particular embodiment of themethods. For example, one rat model of neuropathic pain involvesdevelopment of tactile allodynia (exaggerated response to otherwiseinnocuous stimuli) after ligation of lumbar spinal nerves (see, e.g.,Chaplan et al. (1994) J. Neurosci. Methods 53:55-63 and Luo et al.(2001) J. Neurosci. 21:1868-1875). Tactile allodynia, one characteristicfeature of neuropathic pain, can be evaluated (e.g., by evaluating pawwithdrawal threshold in response to application of pressure) andcompared in animals treated and not treated with test agent.

Animal (e.g., rodent) models of neurodegenerative diseases (e.g.Alzheimer's disease) can also be used in a particular embodiment of themethods. Numerous transgenic mice exhibiting various characteristics ofAD and other neurodegenerative diseases are available. These have beenmade using APP, PS1, PS2, Tau, APOE and other genes, alone and incombinations (seewww.alzforum.org/members/resources/transgenic/index.html).Characteristic phenotypes of neurodegenerative disease (e.g., senileplaques, neuritic plaques, and components of each, neurofibrillarytangles, tau protein and abnormal phosphorylation of tau protein,amyloid precursor protein (APP) and processing thereof, Aβ protein, α-,β- and γ-secretases, presenilin proteins, amyloid deposition, memory orlearning deficits (which can be tested in rodents by the Morris watermaze (Stewart and Morris (1993) “Behavioral Neuroscience,” IRL Press, R.Saghal ed. 107) and the Y-maze (Brits et al. (1981) Brain Res. Bull.6:71) can be evaluated and compared in model animals treated with andnot treated with test agent.

3. Evaluation of Models and Effects of Test Agents Thereon

Cell and animal models of altered intracellular calcium (e.g., calciumdysregulation or dyshomeostasis) and/or of diseases and disordersinvolving or characterized at least in part by calcium dyshomeostasis ordysregulation or alterations in intracellular calcium have a number ofuses. For example, by evaluating the cellular or organismal phenotypesassociated with the altered expression of proteins involved inintracellular calcium modulation (and/or with the expression of apolymorphic form of such proteins) in the cells/organisms andcorrelating such phenotypes with specific cellular molecules andprocesses, the disease/disorder models can be used in elucidating themechanisms underlying calcium dyshomeostasis in a cell as well as indissecting processes and pathways involved in intracellular calciumregulation. In addition, by evaluating the effects of test agents orcandidate therapeutic agents on intracellular calcium and the phenotypicmanifestations of the model cells/organisms, the models can be used inscreening agents and testing candidate agents for the treatment ofdiseases and disorders that involve alterations in intracellular calciumor calcium dyshomeostasis.

In particular embodiments of the methods of screening for or identifyingan agent or candidate agent for the treatment or prevention of diseasesand disorders, a model cell and/or organism, or portion(s) thereof, iscontacted with or exposed to a test or candidate agent. The effect ofthe test or candidate agent on (1) intracellular calcium (and, inparticular, store-operated calcium entry, cytosolic calcium buffering,calcium levels in or movement of calcium into, out of or within anintracellular calcium store and/or resting cytosolic calcium levels)and/or (2) a phenotypic manifestation of a disease or disorder, or ofaltered intracellular calcium or calcium dyshomeostasis, is monitored orassessed. A variety of methods may be used for evaluating suchparameters and are described herein and/or known in the art. Theevaluation method used can depend on the aspect of intracellular calciumbeing assessed or the particular phenotype being assessed. In oneembodiment of the methods, the effect of a test agent on intracellularcalcium or a phenotypic manifestation of altered intracellular calciumor a disease/disorder can be assessed by comparing intracellular calciumor phenotypes of a cell or organism that has been exposed to orcontacted or treated with test agent (test cell or organism) and a cellor organism that has not been exposed to or contacted or treated withtest agent (control cell or organism). The control cell or organism canbe, for example, the test cell or organism prior to contact with orexposure to test agent or can be substantially similar to the test cellor organism. In general, a test agent can be identified as an agent orcandidate agent for use in treating or preventing a disease or disorderif the parameter being assessed and compared differs in the test cell ororganism and the control cell or organism.

For example, in some embodiments of the methods, a test or candidateagent that alters intracellular calcium (e.g., calcium levels and/ormovement of calcium into, out of or within a cell) of a model or testcell (or portion thereof) is identified as an agent or candidate agentfor the treatment or prevention of a disease or disorder involving analteration in intracellular calcium or calcium dyshomeostasis. In oneembodiment, the test cell is one that exhibits calcium dyshomeostasis.Thus, for example, in such an embodiment, a test agent may be identifiedas an agent for use in treating or preventing a disease calciumhomeostasis of a test cell is restored in the presence of the test agent(or after exposure to or contact or treatment with the test agent). Inanother embodiment, the test or model cell is one that contains apolymorphic form of a protein (or of a gene, or portion thereof, ornucleic acid encoding a protein) that is involved in modulatingintracellular calcium. In this embodiment, a test agent may beidentified as an agent for use in treating or preventing a disease, forexample, if intracellular calcium of a test cell is altered in thepresence of the test agent (or after exposure to or contact or treatmentwith the test agent) such that there is less of a difference betweenintracellular calcium of the test cell and a cell that contains awild-type or reference form of the protein (or gene or nucleic acid orportion thereof).

In other embodiments of the methods, a test or candidate agent thatalters a phenotype (e.g., a phenotype associated with a disease ordisorder, including, e.g., a disease or disorder that involves or ischaracterized at least in part by altered intracellular calcium orcalcium dyshomeostasis) of a model or test organism is identified as anagent or candidate agent for the treatment or prevention of a disease ordisorder. An alteration or effect on a phenotype can be, for example, atleast a partial reversal or reduction, or an elimination of a diseasetrait or phenotype exhibited by a model or test organism, or a partialor complete restoration of calcium homeostasis or modulation of calciumsignaling or movement to compensate for disease-associated abnormalitiesin intracellular calcium. A disease trait or phenotype can be any one ormore feature(s) of an organism that has a disease or disorder or alteredintracellular calcium which contribute to the characterization of theorganism as differing from an organism that does not have the disease ordisorder or altered intracellular calcium. Thus, there are numerous andvaried disease traits or phenotypes depending on the disease or disorder(e.g., particular clinical, morphological and histological features, andaltered blood pressure, saliva flow rates, allodynia). A test agent canbe identified as an agent or candidate agent for use in treatment of adisease or disorder if it ameliorates or eliminates the symptoms and/ormanifestations of an inherited or acquired disease or disorder in amodel cell and/or organism. A test agent can be identified as an agentor candidate agent for use in preventing or curing the disease ordisorder if it at least partially restores the wild-type phenotype. Thismay include modulation of store-operated calcium entry, restingcytosolic calcium levels, calcium buffering, and/or calcium levels in ormovement of calcium into, out of or within an intracellular calciumstore in model cells and/or organisms. Intracellular calcium or aphenotype of a test or model cell or organism that has been contactedwith or exposed to a test agent can also be compared to intracellularcalcium or a phenotype of a control cell or organism that is considereda normal or non-disease cell or organism.

G. Methods of Treating a Disease or Disorder

Proteins described herein as being involved in modulating intracellularcalcium, as well as agents that modulate intracellular calcium(including agents described herein and that are identified using methodsprovided herein), are useful in elucidating cellular processes forcalcium homeostasis and signaling. The proteins involved in modulating,and the agents that modulate, intracellular calcium provided herein, arealso useful in the development of methods for treating or preventingdiseases and disorders.

Because of the important role that calcium regulation plays in manycellular processes including cellular activation, gene expression,cellular trafficking and apoptotic cell death, calcium dyshomeostasis isimplicated in the many diseases and disorders involving such cellularactivities. In addition, because any of a number of components(including components that are not direct regulators of intracellularcalcium or that are involved downstream of a calcium signal in a cell)of such critical cellular processes can be altered or defective indiseases and disorders, altering of intracellular calcium in cells thathave such defects (but that do not necessarily have calciumdyshomeostasis or dysregulation) can be an effective treatment bycompensating for the defect, counteracting the defect, reversing thedefect, or alleviating or eliminating the defect. Such cells need nothave altered intracellular calcium or calcium dyshomeostasis; yet,because intracellular calcium (and regulation thereof) plays animportant role in such cellular processes, modulating intracellularcalcium of such cells can be an effective treatment or prophylaticmethod. Diseases/disorders that involve defects that may be at leastpartially compensated for by modulation of intracellular calciuminclude, but are not limited to: autoimmune disorders, includingSjogren's syndrome, asthma, glomerulonephritis and glomerularinflammation, neurological, neurodegenerative, immune system-related,inflammatory, liver and kidney diseases and disorders, aging-relateddisorders, and sensitivity to pain and touch.

Provided herein are methods for treating or preventing a disease ordisorder. The disease or disorder can be one that involves, or ischaracterized at least in part by, (1) altered intracellular calcium,altered intracellular calcium regulation or calcium dyshomeostasis ordysregulation or (2) an alteration or defect in, or aberrant functioningof, a cellular process which relies on or is regulated by intracellularcalcium. The disease or disorder can also be any such disease ordisorder that is not cancer or a neoplastic disease. Such methods caninclude a step of therapeutically modulating a protein (and/or a gene ornucleic acid encoding a protein) involved in modulating intracellularcalcium in a subject having such a disease or disorder or at risk ofdeveloping such a disease or disorder. Therapeutic modulation can bemodulation that is associated with an effective reduction, ameliorationor elimination of a symptom or manifestation of the disease or disorderor that cures the disease or disorder. The step can be a step ofspecifically or selectively modulating a particular protein (and/or geneor nucleic acid encoding a particular protein) involved in modulatingintracellular calcium in a subject.

In particular embodiments, the level of, expression of, a molecularinteraction of and/or an activity or function of a protein (and/or gene,or portion thereof, or nucleic acid encoding a protein) involved inmodulating intracellular calcium is modulated in a subject having such adisease or disorder. In one embodiment, the method includes a step ofadministering to a subject having such a disease or disorder, or at riskfor developing such a disease or disorder, an agent that modulates aprotein (and/or a gene or nucleic acid encoding a protein) involved inmodulating intracellular calcium. The agent can be a therapeutic agent,e.g., drug, pharmacological agent, pharmaceutical agent, that is atherapeutically active substance that can be delivered to a livingorganism to produce a desired, usually beneficial, effect. Agentsinclude, but are not limited to, amino acids, peptides, polypeptides,peptiomimetics, nucleotides, nucleic acids (including DNA, cDNA, RNA,antisense RNA, interfering RNA, such as siRNA, and any double- orsingle-stranded forms of nucleic acids and derivatives and structuralanalogs thereof), polynucleotides, saccharides, fatty acids, steroids,carbohydrates, lipids, lipoproteins, glycoproteins, synthetic or naturalchemical compounds, such as simple or complex organic molecules andmetal-containing compounds. The agent can be one that specifically orselectively modulates a protein (and/or a gene or nucleic acid encodinga protein) involved in modulating intracellular calcium. In particularembodiments, the agent modulates the level of, expression of, amolecular interaction of and/or an activity or function of a protein(and/or gene, or portion thereof, or nucleic acid encoding a protein)involved in modulating intracellular calcium. In particular embodiments,the disease or disorder involves altered store-operated calcium entry,altered resting cytosolic calcium levels, altered calcium buffering,and/or altered calcium levels in or movement of calcium into, out of orwithin an intracellular calcium store (e.g., endoplasmic reticulum).

In one embodiment of the methods for treating or preventing a disease ordisorder, the protein that is being modulated, or that is modulated byan agent being administered, is a protein involved in modulatingintracellular calcium provided herein and described above (and elsewhereherein). Thus, for example, the protein can be one that is homologous toan amino acid sequence of the protein encoded by the coding sequence ofDrosophila gene CG9126 and/or to a mammalian stromal interactingmolecule (STIM) protein, e.g., human or rodent (such as rat or hamster)STIM1. In a particular example, the protein can be at least about 45%homologous to a specified protein over at least about 52% of theprotein. In particular embodiments, the protein is one that is involvedin, participates in and/or provides for store-operated calcium entry,movement of calcium into, out of or within an intracellular calciumstore or organelle, modulation of calcium levels in intracellularcalcium stores or organelles, and/or cytosolic calcium buffering. Inparticular embodiments, the protein is one of the proteins (or issubstantially homologous to one of the proteins) listed in Table 3. Theprotein can be, for example, a STIM or STIM-like protein, including aSTIM1, STIM2, DSTIM-like or CSTIM-like protein. In one embodiment of themethods, the protein is a STIM1 protein, for example, a mammalian (suchas human) STIM1 protein.

Methods of modulating the level of, expression of, a molecularinteraction of, an activity of and/or a function of a protein (and/orgene, or portion thereof, or nucleic acid encoding a protein) involvedin modulating intracellular calcium, and, in particular, the proteinsprovided herein above, are described herein. Also, agents that modulatethe level of, expression of, a molecular interaction of, an activity ofand/or a function of a protein (and/or gene, or portion thereof, ornucleic acid encoding a protein) involved in modulating intracellularcalcium, and, in particular, the proteins provided herein above, aredescribed herein and/or can be identified using methods of screening forintracellular calcium-modulating or treatment agents provided herein. Inparticular embodiments, the agent is one that specifically modulates aparticular protein involved in modulating intracellular calcium. Forexample, the agent can be one that specifically or selectively binds toor interacts with a protein involved in modulating intracellular calciumor a gene (or portion thereof) or nucleic acid encoding such a proteinand/or that is based on a specific feature, e.g., amino acid or nucleicacid sequence, of the protein. Included among such agents are compoundsthat specifically or selectively bind to or interact with such aprotein, antibodies that specifically or selectively recognize such aprotein, peptides that specifically alter (e.g., disrupt or inhibit)interaction of such a protein with another molecule, and nucleic acidsthat specifically bind to or hybridize with a gene, or portion thereof,or nucleic acid (e.g., DNA, RNA, mRNA) encoding such a protein.

In one embodiment, the level and/or expression of a protein (and/or geneor nucleic acid encoding a protein) involved in modulating intracellularcalcium can be altered (e.g., increased or reduced) or modulated. Forexample, an agent that modulates (e.g., increases or decreases) thelevel and/or expression of a protein (and/or gene or nucleic acidencoding a protein) involved in modulating intracellular calcium can beadministered to a subject in such methods. In a particular embodiment,the level and/or expression of a protein (and/or gene or nucleic acidencoding a protein) involved in modulating intracellular calcium isspecifically or selectively altered.

In one method, the level of a protein in a cell of a subject can bealtered, for example, by altering the level of expression of nucleicacid encoding the protein. The level of expression of nucleic acidencoding the protein can be specifically or selectively altered. Agentsfor use in altering nucleic acid expression include agents thatfunctionally bind to or otherwise modulate transcription regulatorysequences that are operably linked to nucleic acid encoding a protein.Such agents can be of any physical type, including peptides, compoundsand nucleic acids. Nucleic acid agents can also be used to alter proteinlevels in other ways. Such nucleic acid agents include inhibitorynucleic acids that reduce the level of and/or reduce or prevent thetranslation of mRNA coding for a protein. Such agents include RNA, e.g.,antisense RNA and molecules that can be used in RNA interference-basedprocesses, such as double-stranded RNA (dsRNA) and small interferingRNAs. The level of a protein in a subject can also be altered byaltering, e.g., increasing, the number of copies of nucleic acidencoding the protein present in cells within the subject. Thus, forexample, an agent can be a nucleic acid, e.g., genomic DNA, cDNA orexpression vector, that is introduced into a subject for expression ofthe protein in cells in the subject, thereby increasing the level of theprotein in the subject.

Agents that can affect expression of a nucleic acid encoding particularproteins provided herein are described herein and/or can be identifiedand produced using methods described herein and/or known in the art. Forexample, an agent that modulates transcription regulatory elements of aSTIM1 gene can be identified as an agent that alters the level ofexpression of a reporter molecule operably linked to STIM1 gene promotersequences. In addition, numerous examples of nucleic acids encoding STIMand STIM-like proteins (and portions thereof) are provided herein andcan be used in designing and producing cDNAs, expression vectors,antisense RNAs and dsRNAs (e.g., siRNAs) using methods known in the art.

In another example of modulating a protein involved in modulatingintracellular calcium, an activity of a protein (and/or a gene ornucleic acid encoding a protein) involved in modulating intracellularcalcium can be altered in a subject by methods other than throughaltering protein (and/or nucleic acid) levels. For example, an activityof a protein (and/or gene or nucleic acid encoding a protein) involvedin modulating intracellular calcium can be increased or reduced. Anactivity of a protein (and/or gene or nucleic acid encoding a protein)involved in modulating intracellular calcium can be specifically orselectively altered. In one embodiment, an agent that modulates anactivity of a protein (and/or gene or nucleic acid encoding a protein)involved in modulating intracellular calcium can be administered to asubject. The activity can be one that is involved in modulatingintracellular calcium either directly (e.g., has an immediate directeffect on intracellular calcium) or indirectly (e.g., an upstream eventin a pathway that ultimately affects intracellular calcium). Alterationsin an activity of the protein include, but are not limited to, increasesand decreases in an activity and/or functioning of the protein.Activities of calcium-modulating proteins include, but are not limitedto, calcium transport, calcium binding, other interactions (e.g.,protein-protein interactions) and regulation of calcium-modulatingproteins. Thus, agents that modulate these activities, or any otheractivity involved in intracellular calcium modulation, can be used inthe methods provided herein. For example, antibodies or other proteinsthat specifically bind to the protein and modulate such activities canbe agents used in the methods. An antibody or other protein may, forinstance, bind to a site of a regulatory protein and reduce or eliminateits binding to the protein it regulates, thereby reducing its regulatoryactivity. Antibodies may also specifically disrupt or inhibit any numberof molecular interactions of a protein involved in modulatingintracellular calcium. A peptide based on a particular amino acidsequence of a protein (e.g., the sequence of a protein-proteininteraction domain) can be an agent that may, for example, alter (e.g.disrupt or inhibit) interaction of a protein with a binding orinteraction partner. Examples of a number of domains, includingprotein-protein interaction domains and regulatory domains, of STIMproteins are described herein and with reference to particular STIMamino acid sequences. Agents that modulate particular proteins providedherein can also be identified using methods described herein and/orknown in the art. Such methods include binding and interaction assaysand assays for detecting disruption or inhibition of binding (e.g.,STIM1 homotypic and STIM1/STIM2 hetero-oligomeric interactions)described above.

Antibody-based agents include antibodies (e.g., monoclonal andpolyclonal) and fragments of antibodies, e.g., an antigen-bindingportion. Fragments include fragments that retain the ability tospecifically bind to an antigen, such as, for example, Fab, F(ab′)₂, Fd,Fv, dAb and CDR (complementarity determining region) fragments. Methodsof producing antibodies and fragments thereof are known in the art.Particular antibodies for use in the methods include antibodies specificfor a STIM or STIM-like protein (or a portion thereof), such as a STIM1or STIM2 protein or portion thereof (including antibodies describedherein). In particular embodiments, an agent for use in the method is anantibody, or fragment thereof, specific for STIM1, such as human STIM1,or a portion thereof. Human monoclonal antibodies directed against humanproteins can be generated using transgenic mice whose genomes includethe human immunoglobulin loci instead of the murine loci. Splenocytesfrom these transgenic mice immunized with the antigen of interest areused to produce hybridomas that secrete human monoclonal antibodies withspecific affinities for epitopes from a human protein. Mouse (or otheranimal) antibodies and chimeric (e.g., mouse-human) antibodies can alsobe humanized using methods known in the art. Such methods includemethods in which at least a portion of a CDR of a human antibody isreplaced with a CDR derived from a non-human antibody.

Peptide or protein fragment agents, e.g., peptides or fragments based ona particular amino acid sequence of a protein (e.g., the sequence of aprotein-protein interaction domain) can also be generated usingtechniques known in the art. Such agents include peptides andbiologically active analogs thereof. Biologically active analogs arepeptide analogs that retain the ability to modulate a protein involvedin modulating intracellular calcium. Examples of peptide analogs arepeptides modified to increase peptide stability (e.g., modified with anon-peptide bond, D-amino acids or non-naturally occurring amino acids)and to make the peptide non-hydrolyzable (through, e.g., peptidebackbone modifications). Peptides and protein fragments can be producedby a variety of methods known in the art, including, for example,recombinantly, by proteolytic digestion, or by chemical synthesis.

In particular embodiments of the methods for treating diseases anddisorders provided herein, the disease or disorder can be, for example,a neurodegenerative disease or disorder, e.g., Alzheimer's disease (AD),Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), and other brain disorders caused by trauma or other insultsincluding aging, an immune system-related disease (e.g., an autoimmunedisease or an immunodeficiency disorder or disease), a disease ordisorder involving inflammation (e.g., asthma, chronic obstructivepulmonary disease, rheumatoid arthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases, multiple sclerosis, anddisorders of the immune system), cancer or other proliferative disease,kidney disease and liver disease.

1. Diseases

a. Neurodegenerative Diseases and Disorders

Diseases or disorders that can be treated or prevented using the methodsprovided herein include neurodegenerative diseases and disorders. Inparticular embodiments, the neurodegenerative disease or disorder is nota cancer or neoplastic disease or disorder. Neurodegenerative diseasesand disorders include but are not limited to Alzheimer's disease (AD),Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), and other brain disorders caused by trauma or other insultsincluding aging.

Mechanisms associated with calcium signaling may be altered in manyneurodegenerative diseases and in disorders resulting from brain injury.For example, fibroblasts or T-lymphocytes from patients with AD haveconsistently displayed an increase in Ca²⁺ release from intracellularstores compared to controls (Ito et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:534-538; Gibson et al. (1996) Biochem. Biophys. ACTA1316:71-77; Etchenberrigaray et al. (1998) Neurobiology of Disease,5:37-45). Consistent with these observations, mutations in presenilingenes (PS1 or PS2) associated with familial AD (FAD) have been shown toincrease IP₃-mediated Ca²⁺ release from internal stores (Guo et al.(1996) Neuro Report, 8:379-383; Leissring et al. (1999) J.Neurochemistry, 72:1061-1068; Leissring et al. (1999) J. Biol. Chem.274(46):32535-32538; Leissring et al. (2000) J. Biol. Chem.149(4):793-797; Leissring et al. (2000) Proc. Natl. Acad. Sci. U.S.A.97(15):8590-8593). Furthermore, mutations in PS1 or PS2 associated withan increase in amyloidogenic amyloid β peptide generation in AD arereported to be associated with a decrease in store-operated calciumentry (Yoo et al. (2000) Neuron, 27(3):561-572).

Experimental traumatic brain injury has been shown to initiate massivedisturbances in Ca²⁺ concentrations in the brain that may contribute tofurther neuronal damage. Intracellular Ca²⁺ may be elevated by manydifferent ion channels including store-operated channels. It has beenfurther shown that channel blockers may be beneficial in the treatmentof neurological motor dysfunction when administered in the acuteposttraumatic period (Cheney et al. (2000) J. Neurotrauma, 17(1):83-91).

b. Diseases/Disorders Involving Inflammation and Diseases/DisordersRelated to the Immune System

Diseases or disorders that can be treated or prevented using the methodsprovided herein include diseases and disorders involving inflammationand/or that are related to the immune system. In particular embodiments,the disease or disorder involving inflammation or the immune system isnot a cancer or neoplastic disease or disorder. These diseases includebut are not limited to asthma, chronic obstructive pulmonary disease,rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis,neuroinflammatory diseases such as multiple sclerosis, and disorders ofthe immune system.

The activation of neutrophils (PMN) by inflammatory mediators is partlyachieved by increasing cytosolic calcium concentration ((Ca²⁺)i).Store-operated calcium influx in particular is thought to play animportant role in PMN activation. It has been shown that traumaincreases PMN store-operated calcium influx (Hauser et al. (2000) J.Trauma Injury Infection and Critical Care 48 (4):592-598) and thatprolonged elevations of (Ca²)i due to enhanced store-operated calciuminflux may alter stimulus-response coupling to chemotaxins andcontribute to PMN dysfunction after injury. Modulation of PMN (Ca²⁺)ithrough store-operated calcium channels might therefore be useful inregulating PMN-mediated inflammation and spare cardiovascular functionafter injury, shock or sepsis (Hauser et al. (2001) J. Leukocyte Biology69 (1):63-68).

Calcium plays a critical role in lymphocyte activation. Activation oflymphocytes, e.g., by antigen stimulation, results in rapid increases inintracellular free calcium concentrations and activation oftranscription factors, including nuclear factor of activated T cells(NFAT), NF-kappaB, JNK1, MEF2 and CREB. NFAT is a key transcriptionalregulator of the IL-2 (and other cytokine) genes (see, e.g. Lewis (2001)Annu. Rev. Immunol 19:497-521). A sustained elevation of intracellularcalcium level is required to keep NFAT in a transcriptionally activestate, and is dependent on store-operated calcium entry. Reduction orblocking of store-operated calcium entry in lymphocytes blockscalcium-dependent lymphocyte activation. Thus, modulation ofintracellular calcium, and particularly store-operated calcium entry(e.g., reduction in, elimination of and enhancement of store-operatedcalcium entry), in lymphocytes can be a method for treating immune andimmune-related disorders, including, for example, chronic immunediseases/disorders, acute immune diseases/disorders, autoimmune andimmunodeficiency diseases/disorders, diseases/disorders involvinginflammation, organ transplant graft rejections and graft-versus-hostdisease and altered (e.g., hyperactive) immune responses. For exampletreatment of an automimmune disease/disorder might involve reducing,blocking or eliminating store-operated calcium entry in lymphocytes.Treatment of an immunodeficiency might involve enhancing store-operatedcalcium entry in lymphocytes. Examples of immune disorders includeimmunodeficiency or severe-combined immunodeficiency (SCID), psoriasis,rheumatoid arthritis, vasculitis, inflammatory bowel disease,dermatitis, osteoarthritis, asthma, inflammatory muscle disease,allergic rhinitis, vaginitis, interstitial cystitis, scleroderma,osteoporosis, eczema, allogeneic or xenogeneic transplantation (organ,bone marrow, stem cells and other cells and tissues) graft rejection,graft-versus-host disease, lupus erythematosus, inflammatory disease,type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren'ssyndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis),myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis,cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis,allergic conjunctivitis and atopic dermatitis.

In a particular embodiment of the methods for treating or preventing adisease/disorder provided herein, the disease or disorder is animmunodeficiency, such as a primary immunodeficiency, T-cellimmunodeficiency or severe combined immunodeficiency (SCID). In oneembodiment, the immunodeficiency is one characterized by a defect incalcium influx, and, in particular, store-operated calcium entry, intolymphocytes, such as T lymphocytes (see, e.g., Feske et al. (2001)Nature Immunol. 2:316-324; Partiseti et al. (1994) J. Biol. Chem.269:32327-32335; and Le Deist et al. (1995) Blood 85:1053-1062). Themethod includes a step of modulating in a cell (or portion thereof) of asubject having such an immunodeficiency (or at risk for developing suchan immunodeficiency), a protein (and/or gene or nucleic acid encoding aprotein) involved in modulating intracellular calcium. The protein canbe one that is homologous to an amino acid sequence of the proteinencoded by the coding sequence of Drosophila gene CG9126 and/or to amammalian stromal interacting molecule (STIM) protein, e.g., human orrodent (such as rat) STIM1. In a particular example, the protein can beat least about 45% homologous to a specified protein over at least about52% of the protein. In particular embodiments, the protein is one thatis involved in, participates in and/or provides for store-operatedcalcium entry, movement of calcium into, out of or within anintracellular calcium store or organelle, modulation of calcium levelsin intracellular calcium stores or organelles, and/or cytosolic calciumbuffering. In particular embodiments, the protein is one of the proteins(or is substantially homologous to one of the proteins) listed in Table3. The protein can be, for example, a STIM or STIM-like protein,including a STIM1, STIM2, DSTIM-like or CSTIM-like protein. In oneembodiment of the methods, the protein is a STIM1 protein. Inparticular, the protein is a human protein.

In one embodiment, the protein (or gene or nucleic acid encoding theprotein) is specifically or selectively modulated in a cell of a subjecthaving an immunodeficiency or at risk for developing such animmunodeficiency. In a particular embodiment, the cell is an immunecell, such as a lymphocyte, e.g., a T lymphocyte. The protein can bemodulated in a number of ways as described herein. For example, thelevel of, expression of, a molecular interaction of and/or an activityor function of the protein (and/or gene, or portion thereof, or nucleicacid encoding a protein) involved in modulating intracellular calciumcan be modulated. The step of modulating the protein can includeadministering to a subject an agent that modulates the protein. Theagent can be one that specifically or selectively modulates the protein.Agents include, for example, but are not limited to, a peptide orpolypeptide having an amino acid sequence of all or part of the protein,a nucleic acid having a nucleotide sequence of, or complementary to, asequence in a gene or nucleic acid (e.g., cDNA or RNA transcript)encoding the protein, and a composition that modulates expression of agene encoding the protein and/or an intracellular calcium-modulatingactivity of the protein. Examples of nucleotide and amino acid sequencesare provided herein (see, e.g., Table 3 and listed SEQ ID NOS.). Agentsthat modulate expression of a gene encoding the protein and/or anintracellular calcium-modulating activity of the protein can beidentified using methods described herein. In a particular embodiment,the method of treating or preventing an immunodeficiency involvesadministering to a subject having an immunodeficiency (or at risk fordeveloping an immunodeficiency) the particular protein involved inmodulating intracellular calcium, a nucleic acid encoding a proteininvolved in modulating intracellular calcium, and/or an agent thatup-regulates or enhances expression of a gene encoding the protein,thereby increasing the level and/or activity of the protein in a cell orcells of the subject. Procedures and reagents for use in delivering suchagents to a subject are described herein and/or known in the art.

C. Cancer and Other Proliferative Diseases

Methods provided herein may also be used in connection with treatment ofmalignancies, including, but not limited to, malignancies oflymphoreticular origin, bladder cancer, breast cancer, colon cancer,endometrial cancer, head and neck cancer, lung cancer, melanoma, ovariancancer, prostate cancer and rectal cancer. Store-operated calcium entrymay play an important role in cell proliferation in cancer cells (Weisset al. (2001) International Journal of Cancer 92 (6):877-882).

d. Liver Diseases and Disorders

Diseases or disorders that can be treated or prevented using the methodsprovided herein include hepatic or liver diseases and disorders. Inparticular embodiments, the hepatic or liver disease or disorder is nota cancer or neoplastic disease or disorder. These diseases and disordersinclude but are not limited to alcoholic liver disease, liver injury,for example, due to transplantation, hepatitis and cirrhosis.

Store-operated calcium entry has been implicated in chronic liverdisease (Tao et al. (1999) J. Biol. Chem., 274(34):23761-23769) as wellas transplantation injury after cold preservation-warm reoxygenation(Elimadi et al. (2001) Am J. Physiology, 281(3 Part 1):G809-G815.Chronic ethanol consumption has been shown to impair liver regeneration,in part, by modulating store-operated calcium entry (Zhang et al. (1996)J. Clin. Invest. 98(5):1237-1244).

e. Kidney Diseases and Disorders

Diseases or disorders that can be treated or prevented using the methodsprovided herein include kidney or renal diseases and disorders. Inparticular embodiments, the kidney or renal disease or disorder is not acancer or neoplastic disease or disorder. Mesangial cell hyperplasia isoften a key feature of such diseases and disorders. Such diseases anddisorders may be caused by immunological or other mechanisms of injury,including IgAN, membranoproliferative glomerulonephritis or lupusnephritis. Imbalances in the control of mesangial cell replication alsoappear to play a key role in the pathogenesis of progressive renalfailure.

The turnover of mesangial cells in normal adult kidney is very low witha renewal rate of less than 1%. A prominent feature of glomerular/kidneydiseases is mesangial hyperplasia due to elevated proliferation rate orreduced cell loss of mesangial cells. When mesangial cell proliferationis induced without cell loss, for example due to mitogenic stimulation,mesangioproliferative glomerulonephritis can result. Data have indicatedthat regulators of mesangial cell growth, particularly growth factors,may act by regulating store-operated calcium channels (Ma et al. (2001)J Am. Soc. of Nephrology, 12:(1) 47-53). Modulators of store-operatedcalcium influx may aid in the treatment of glomerular diseases byinhibiting mesangial cell proliferation.

2. Agents for Treatment

Agents for use in the methods of treating a disease or disorder can beany substance or combination of substances that modulates the level of,expression of, a molecular interaction of and/or an activity of aprotein involved in modulating intracellular calcium as provided herein.Examples of agents include, but are not limited to, chemical compounds,small organic molecules, amino acids, peptides, polypeptides,nucleotides, nucleic acids, polynucleotides, carbohydrates, lipids,lipoproteins and glycoproteins. Generally, an agent for treatment of adisease or disorder is a composition, such as a compound or combinationof compounds, that when administered to a subject having a disease ordisorder effectively reduces, ameliorates or eliminates a symptom ormanifestation of the disease or disorder or that cures the disease ordisorder. An agent can also be a composition that, when administered toa subject predisposed to a disease or disorder who does not yet manifesta symptom of the disease or disorder, prevents or delays development ofthe symptoms. The agent can have such effects alone or in combinationwith other agents, or may function to enhance a therapeutic effect ofanother agent.

3. Methods of Delivering an Agent for Treatment

Agents for use in the methods of treating or preventing a disease ordisorder as provided herein may be delivered to a subject using anymethods known in the art or described herein, particularly methods thatprovide for delivery of agent to target cells in which intracellularcalcium may be altered. Typically, delivery of an agent involves theadministration of an effective amount (e.g., a therapeutically effectiveamount) of agent or a pharmaceutically acceptable salt or derivativesthereof. The agent may be administered with a pharmaceuticallyacceptable, non-toxic, excipient, including solid, semi-solid, liquid oraerosol dosage forms.

Administration of the agent can be via a variety of modes andformulations for administering compounds to subjects. For example, theagent may be administered orally, by injection (e.g., intravenous,intradermal, subcutaneous, intramuscular or intraperitoneal), byinhalation, nasally, intrabronchially, rectally, parenterally,intravascularly, transdermally (including electrotransport), byimplantation (e.g., insertion of implantable drug delivery systems suchas microspheres, hydrogels, polymeric reservoirs, cholesterol matrices,polymeric systems and non-polymeric systems) or topically, in the formof a solid, semi-solid, lyophilized powder, or liquid dosage forms, suchas, for example, tablets, suppositories, pills, capsules, powders,solutions, suspensions, emulsions, creams, lotions, aerosols, ointments,injectables and gels, preferably in unit dosage forms suitable forsimple administration of precise dosages. The compositions can include apharmaceutical carrier or excipient and an active compound (i.e., theagent or pharmaceutically acceptable salt or derivative thereof), and,in addition, may include other medicinal agents, pharmaceutical agentcarriers, adjuvants and other such substances.

For oral administration, a pharmaceutically acceptable, non-toxiccomposition may be formed by the incorporation of excipients, such as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,gelatin, sucrose and magnesium carbonate. Such compositions may takeseveral forms, such as solutions, suspensions, tablets, pills, capsules,powders and sustained release formulations. The composition may contain,along with the active ingredient, a diluent, such as lactose, sucrose,dicalcium phosphate, a disintegrant, such as starch or derivativesthereof, a lubricant, such as magnesium stearate, and a binder, such asstarch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose andderivatives thereof.

Liquid formulations may, for example, be prepared by dissolving,dispersing an active agent or compound (for example, about 0.1% to about95%, or 0.1% to about 50%, or about 0.5% to about 20%) and optionalpharmaceutical adjuvants in a carrier, such as water, saline, aqueousdextrose, glycerol, and ethanol, to thereby form a solution orsuspension. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art; for example, seeRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 15th ed., 1975.

For parenteral administration, the agent may be mixed with a carrier,such as, for example, an oily ester such as ethyl oleate and isopropylmyristate. Sterile liquid pharmaceutical compositions, solutions orsuspensions can be utilized by, for example, intraperitoneal injection,subcutaneous injection, intramuscular injection or intravenously.

Transdermal administration of the agent may be conducted through the useof a patch containing the agent and a carrier that is inert to theagent, is non-toxic to the skin and allows delivery of the agent forsystemic absorption into the blood stream via the skin. Carriers fortransdermal absorption may include pastes, e.g., absorptive powdersdispersed in petroleum or hydrophilic petroleum containing the agentwith or without a carrier or a matrix containing the agent; creams andointments, e.g., viscous liquid or semi-solid emulsions, gels andocclusive devices.

Generally, an agent is administered to achieve an amount effective foramelioration of, or prevention of the development of symptoms of, thedisease or disorder (i.e., a therapeutically effective amount). Thus, atherapeutically effective amount can be an amount which is capable of atleast partially preventing or reversing a disease or disorder. The doserequired to obtain an effective amount may vary depending on the agent,formulation, disease or disorder, and individual to whom the agent isadministered.

Determination of effective amounts may also involve in vitro assays inwhich varying doses of agent are administered to cells in culture andthe concentration of agent effective for ameliorating some or allsymptoms is determined in order to calculate the concentration requiredin vivo. Effective amounts may also be based in in vivo animal studies.A therapeutically effective amount can be determined empirically bythose of skill in the art.

An agent can be administered prior to, concurrently with and subsequentto the appearance of symptoms of a disease or disorder. In particularembodiments, an agent is administered to a subject with a family historyof the disease or disorder, or who has a phenotype that may indicate apredisposition to a disease or disorder, or who has a genotype whichpredisposes the subject to the disease or disorder.

The particular delivery system used can depend on a number of factors,including, for example, the intended target and the route ofadministration, e.g., local or systemic. Targets for delivery can bespecific cells which are causing or contributing to a disease ordisorder, including, for example, cells that have altered intracellularcalcium or calcium dysregulation or dyshomeostasis, and cells that donot have altered intracellular calcium but that may have somealteration, defect or deficiency that can be, at least in part,compensated, counteracted, reversed or alleviated or eliminated byaltering intracellular calcium of the cell. Particular cells include,for example, immune cells (e.g., lymphocytes, T cells, B cells, whiteblood cells), fibroblasts (or cells derived from a fibroblast),epidermal, dermal or skin cells (e.g., a keratinocytes), blood cells,kidney or renal cells (e.g., mesangial cells), muscle cells (e.g., asmooth muscle cell such as an airway (tracheal or bronchial) smoothmuscle cell) and exocrine or secretory (e.g., salivary, includingparotid acinar and submandibular gland) cells. For example, a targetcell can be resident or infiltrating cells in the lungs or airways thatcontribute to an asthmatic illness or disease, resident or infiltratingcells in the nervous system contributing to a neurological,neurodegenerative or demyelinating disease or disorder, resident orinfiltrating cells involved in rejection of a kidney graft, graftedcells that when activated lead to graft-versus-host disease, resident orinfiltrating cells involved in rejection of a kidney graft, resident orinfiltrating cells, activation of which contributes to inflammation,e.g., in arthritis, resident or infiltrating cells in the kidney orrenal system (e.g., mesangial cells) involved in neuropathy andglomerulonephritis and resident or infiltrating cells in exocrine glands(e.g., salivary and lacrimal glands) involved in autoimmune disorders(e.g., Sjogren's disease). Administration of an agent can be directed toone or more cell types or subsets of a cell type by methods known tothose of skill in the art. For example, an agent can be coupled to anantibody, ligand to a cell surface receptor or a toxin, or can becontained in a particle that is selectively internalized into cells,e.g., liposomes or a virus in which the viral receptor bindsspecifically to a certain cell type, or a viral particle lacking theviral nucleic acid, or can be administered locally.

Nucleic acid agents (including RNA and DNA) for use in the methods canbe delivered to a subject in a number of ways known in the art,including through the use of gene therapy vectors and methods. If thenucleic acid is one to be expressed in the subject, it can be operablylinked to transcription regulatory nucleotide sequences such as promoterelements and 5′ and 3′ untranslated sequences. Regulatory sequences canbe those naturally associated in a genome with the expression of thenucleic acid sequence or can be from another gene. For example, it maybe desired to provide for a particular type of inducible expression ofthe nucleic acid in a subject or a tissue- or cell-specific expressionthat is other than the natural expression pattern of the nucleic acid,in which case regulatory elements from another gene may be operablylinked to the nucleic acid. Nucleic acids that can be expressed in asubject include nucleic acids that encode a protein, peptide and/or anRNA molecule. RNA molecules include inhibitory molecules. For example,small interfering RNA molecules (siRNA) can be produced within a cellas, for example, hairpin transcripts (short hairpins or shRNA) (see,e.g., Qin et al. (2003) Proc. Natl. Acad. Sci. 100:183-188; Tiscornia etal. (2003) Proc. Natl. Acad. Sci. 100:1844-1848; and Robinson et al.(Feb. 18, 2003) Nature Genet. doi:10.1038/ng117). The nucleic acid canbe contained within a vector that can be one useful in gene therapy, forexample, a vector that can be transferred to the cells of a subject andprovide for expression of the therapeutic nucleic acid agent therein.Such vectors include chromosomal vectors (e.g., artificial chromosomes),non-chromosomal vectors and synthetic nucleic acids. Vectors includeplasmids, viruses and phage, such as retroviral vectors, lentiviralvectors, adenoviral vectors and adeno-associated vectors.

Nucleic acid agents can be transferred into a subject using ex vivo orin vivo methods. Ex vivo methods involve transfer of the nucleic acidinto cells in vitro (e.g., by transfection, infection or injection) thatare then transferred into or administered to the subject. The cells canbe, for example, cells derived from the subject (e.g., lymphocytes) orallogeneic cells. For example, the cells can be implanted directly in toa specific tissue of the subject or implanted after encapsulation withinan artificial polymer matrix. Examples of sites of implantation includethe lungs or airways, skin, conjunctiva, central nervous system,peripheral nerve, a grafted kidney or an inflammed joint. Nucleic acidscan also be delivered into a subject in vivo. For example, nucleic acidscan be administered in an effective carrier, e.g., any formulation orcomposition capable of effectively delivering the nucleic acid to cellsin vivo. Nucleic acids contained within viral vectors can be deliveredto cells in vivo by infection or transduction using virus. Nucleic acidsand vectors can also be delivered to cells by physical means, e.g., byelectroporation, lipids, cationic lipids, liposomes, DNA gun, calciumphosphate precipitation, injection or delivery of naked nucleic acid.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

H. EXAMPLES Example 1

Evaluation of CG9126 Gene in Intracellular Calcium Modulation

This Example describes procedures used to silence the CG9126 Drosophilagene in S2 cells using RNA interference (RNAi) and to assess the effectsof CG9126 gene silencing on basal cytosolic calcium levels andstore-operated calcium entry. The results obtained are consistent withthe CG9126 gene product being involved in modulating intracellularcalcium.

A. RNAi Protocol

1. Cell Culture

S2 cells (Invitrogen) were propagated in Drosophila expression system(DES) media (Invitrogen) supplemented with 12.5% FBS, and 200 U/Lpenicillin/streptomycin (DES complete media). Cells were plated at adensity of 1×106 cells/ml until they became ˜90% confluent in a 75-cm2T-flask. Cells were incubated at 22° C.

2. Double-Stranded RNA (dsRNA) Production

PCR primers were designed based on the CG9126 coding sequence (GenBankAccession No. AF328906). CG9126 DNA was amplified from S2 cells usingthe EXPAND Long Template PCR system (Roche) as per manufacturer'sprotocols with the following primers (both internal designations andsequence identifiers are given for each primer).

The sense strand primers contained a 5′ T7 RNA polymerase binding site(GAATTAATACGACTCACTATAGGGAGA; SEQ ID NO: 47) followed by a sequencespecific for the CG9126 gene.

Sense-Strand Primers: Dm000223U2 (SEQ ID NO: 15) Dm000223U55 (SEQ ID NO:17) Dm000223U3 (SEQ ID NO: 19) Dm000223U4 (SEQ ID NO: 21)The antisense-strand primers contained a 5′ T3 RNA polymerase bindingsite (AATTAACCCTCACTAAAGGGAGA; SEQ ID NO: 48) followed by sequencespecific for the CG9126 gene.

Antisense-Strand Primers: Dm000223L2 (SEQ ID NO: 16) Dm000223L556 (SEQID NO: 18) Dm000223L3 (SEQ ID NO: 20) Dm000223L4 (SEQ ID NO: 22)

Double-stranded RNA (dsRNA) was generated from the PCR amplificationproducts (SEQ ID Nos. 23 and 24) using the T7 Megascript and T3Megascript Kits (Ambion, Austin, Tex.). Duplex RNA was achieved byheating equivalent amounts of sense and antisense stands to 65° C. for30 minutes and then slow cooling to room temperature. The concentrationof dsRNA was determined by spectrophotometry (optical density at 260nm). Double-stranded RNA was stored at −20° C.

3. Conditions for RNAi in Drosophila Cell Culture

Drosophila S2 cells were diluted to a final concentration of 1×106cells/ml in DES complete medium. Six milliliters of cells were plated ina 75 cm2 T-flask. The cells were allowed to attach to the flask, and theDES complete media was replaced with 6 ml DES serum-free media. dsRNAwas added directly to the media to an approximate final concentration of37 nM and mixed by agitation. The cells were incubated for 30 min atroom temperature followed by addition of 12 ml of DES complete media.The cells were treated with the dsRNA on day 0 of the time course. Thecells were incubated and analyzed over a 4-day period to determineturnover of the target gene.

B. Analysis of CG9126 Gene Silencing

To confirm dsRNA-mediated silencing of CG9126, RNA was isolated from theS2 cells using an RNeasy™ kit from Qiagen, and RT-PCR used to monitorCG9126 mRNA levels. A reduction in CG9126 mRNA levels in cells treatedwith CG9126 dsRNA relative to control cells was observed starting onday 1. By day 2, CG9126 mRNA levels in cells treated with CG9126 dsRNAwere reduced to less than 10% of control cells. CG9126 mRNA levels indsRNA-treated cells remained at less than 10% of control cells on days 3and 4 of the time course analysis.

C. Measurement of Cytosolic Calcium

Control S2 cells and S2 cells subjected to RNAi for the silencing ofCG9126 were analyzed for possible effects of gene silencing on cytosoliccalcium using a fluorescence-based assay.

1. Fluorescence-Based Assay Protocol

S2 cells, and S2 cells treated with CG9126 dsRNA, as described above,were seeded in a 96-well plate at a density of 100,000 cells/well (100μl of 106 cells/ml stock). The following day, the media was removed andcells were washed with 2 mM Ca²⁺ buffer (120 mM NaCl, 5 mM KCl, 4 mMMgCl.6H₂O, 2 mM CaCl₂.2H₂O, 10 mM HEPES, pH 7.4). Cells were loaded with50 μl of 10 μM FLUO-4-AM dye (a Ca²⁺-sensitive fluorescent dye) in 2 mMCa²⁺ buffer supplemented with 2.5 mM probenecid and incubated for 60minutes at room temperature in the dark. The dye was removed and cellswere washed with nominally Ca²⁺-free buffer (120 mM NaCl, 5 mM KCl, 4 mMMgCl.6H2O, 10 mM HEPES, pH 7.4). Cells were then incubated in 90 μl Ca²⁺free buffer supplemented with 2.5 mM probenecid. Probenecid inhibits themultidrug resistance transporter, which otherwise would transport loadeddye out of the cell.

Fluorescence of the dye-loaded cells was measured using an excitationwavelength of 485 nm and an emission wavelength of 510 nm on aFluoroSkan Ascent fluorimeter (Lab Systems). Both an Fmax and cellularfluorescence were measured for each well. The Fmax is the maximumfluorescence of FLUO-4, derived after lysis of cells, which is measured10 minutes after adding 10 μl of 1% Triton X-100 to each well at the endof the experiment. The Fmax provides a measure of the relative amount ofdye loaded in each well of the 96-well plate, and is proportional to thetotal number of cells in each well. By taking Fmax into account, anydifferences in fluorescence of different wells due to well-to-wellvariability that may arise during the plating procedure can be factoredout of the analysis. Cellular fluorescence is the FLUO-4-dependentfluorescence from intact cells of a well.

2. Evaluation of Basal Cytosolic Calcium

Initial basal, resting calcium levels were determined in the absence ofexternal Ca²⁺ by measuring the fluorescence of the wells prior to addingany type of agent (e.g., store-depletion agent) or manipulating themedium (e.g., altering the calcium concentration of the medium) in orderto evaluate store-operated calcium entry as described below. Todetermine basal calcium levels, the cellular fluorescence of a well wasdivided by the Fmax for that well at the beginning of each experiment.The cellular fluorescence/Fmax values of different wells containingcontrol cells and cells subjected to RNAi to silence CG9126 geneexpression were compared.

The results of these comparisons revealed that the basal, restingcytosolic calcium levels of S2 cells on day 4 after treatment with dsRNAcorresponding to CG9126 gene sequence was nearly the same as the restingcytosolic calcium levels of control cells.

3. Evaluation of Store-Operated Calcium Entry

Store-operated calcium channels have specific pharmacologicalcharacteristics that are distinct from other ion channels.Store-operated calcium channels are activated in response to depletionof intracellular calcium stores, which is one feature that distinguishesthem from other ion channels such as voltage-gated channels.Store-operated calcium entry can thus be monitored by using storedepletion to activate channel activity. Either passive or active storedepletion may be used to activate store-operated channel activity.Passive intracellular store depletion can be achieved using a bufferingagent such as EGTA or chelator such as TPEN. Active intracellular Ca²⁺store depletion can be achieved using inositol-1,4,5-triphosphate(InsP3) to release Ca²⁺ from the stores directly, or by using an agonistthat activates a receptor leading to an increase in InsP3. Thapsigarginor thapsigargin-like compounds and compounds such as ionomycin, BHQ, andcyclopiazonic acid may also be used to actively deplete Ca²⁺ fromintracellular stores. Thapsigargin (Tg) acts to inhibit the ER Ca²⁺ pumpand discharge intracellular Ca²⁺ stores. In the absence of externalCa²⁺, thapsigargin elicits a transient elevation in cytosolic free Ca²⁺concentration ([Ca²⁺]i), which is believed to result from release ofstored Ca²⁺, followed by removal or buffering of cytosolic Ca²⁺ back toprestimulation levels. Following measurement of basal calcium, S2 cellswere incubated with either 10 μl of 0.1% DMSO (control) or 10 μl of 10μM thapsigargin (final concentration of 1 μM) for 5 minutes at roomtemperature. Ten microliters of 20 mM Ca²⁺ buffer (120 mM NaCl, 5 mMKCl, 4 mM MgCl.6H2O, 20 mM CaCl₂.2H2O, 10 mM HEPES, pH 7.4) was thenadded to the cells, and the cells were incubated for 3 minutes at roomtemperature. Cytosolic calcium levels were then evaluated by measuringfluorescence of the wells.

To evaluate any cytosolic calcium level increases due to store-operatedentry of calcium into the cell from the extracellular mediumindependently of any increased cytosolic calcium levels induced by nonstore-operated means, the basal fluorescence (measured as describedabove) was subtracted from the cellular fluorescence measured aftertreatment with or without thapsigargin and incubation incalcium-containing medium. The adjusted fluorescence value was thendivided by Fmax for the particular well. These manipulations distinguishan effect on non store-operated calcium from an effect on store-operatedcalcium entry (i.e., thapsigargin-dependent calcium entry).

Analysis of these fluorescence values revealed thatthapsigargin-dependent entry of calcium was significantly reduced in S2cells that had been subjected to RNAi-mediated silencing of CG9126 genecompared to control cells.

Example 2

Evaluation of STIM1 in Intracellular Calcium Modulation

This Example describes procedures used to silence the STIM1 gene inhuman and rodent cell lines using RNA interference (RNAi) and todetermine the effects of STIM1 gene silencing on thapsigargin-inducedand agonist-induced store-operated calcium entry. The results obtainedare consistent with STIM1 being involved in modulating intracellularcalcium.

A. RNAi Protocol

1. Cell Culture

HEK293 human embryonic kidney cells were propagated in HEK completemedia (90% DMEM, 10% FBS, 2 mM L-glutamine, 1.5 mg/l sodium bicarbonate,0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate). SH-SY5Yhuman neuroblastoma cells were propagated in SY5Y complete media (45%DMEM without L-glutamine, 45% F-12 medium, 10% FBS, 10 ml sodiumpyruvate, 10 ml Pen-strep glutamine, 1.5 mg/l sodium bicarbonate and 0.1mM non-essential amino acids). Chinese hamster ovary (CHO) cells werepropagated in 85% DMEM without L-glutamine, 15% FBS, 10 ml Pen-strepglutamine, 1.5 mg/l sodium bicarbonate and 0.1 mM non-essential aminoacids.

2. Small Interfering RNA (siRNA) Production

To design STIM siRNAs, STIM1 and STIM2 cDNA sequences were examined forAA(N19)TT sequences with a GC content of about 45-60%, and without astretch of three G nucleotides in either the sense or antisense strand.Based on these parameters, two human STIM1 siRNAs were synthesized(Dharmacon Research, Lafayette, Colo.), with sense strands correspondingto nucleotides 1140-1160 (huSTIM1-1140; SEQ ID NO:43) and 1414-1434(huSTIM1-1414; SEQ ID NO:44). Two human STIM2 siRNAs were alsosynthesized, with sense strands corresponding to nucleotides 1550-1570(huSTIM2-1550; SEQ ID NO:45) and 2560-2580 (huSTIM2-2560; SEQ ID NO:46),as well as two hamster STIM1 siRNAs, with sense strands corresponding tonucleotides 114-134 (rSTIM1-114; SEQ ID NO:91) and nucleotides 614-634(rSTIM1-614; SEQ ID NO:92).

3. Conditions for RNAi in Mammalian Cell Culture

For experiments in 6-well dishes, cells were plated on poly-D-lysine(PDL)-coated dishes at the following densities: HEK293 cells at 300,000cells/well, SH-SY5Y cells at 700,000 cells/well, and CHO cells at300,000 cells/well on a PDL coated 6-well dish. For experiments in T-75flasks, ten times as many cells were plated.

On day 0, cells were transfected with siRNA using the TransMessengerTransfection Reagent™ kit (Qiagen) according to the following procedure.Four microliters of Enhancer R (Qiagen) were diluted with 90 EC-R buffer(Qiagen), and 6.7 μl of siRNA (0.3 mg/ml; 20 μM) was added. The solutionwas mixed by pipetting or briefly vortexing, and then incubated 5minutes at room temperature. Eight microliters of TransMessenger(Qiagen) was then added. The solution was mixed by pipetting or brieflyvortexing, and then incubated a further 10 minutes at room temperature.While the transfection complexes were forming, the cells were washedwith PBS. Nine-hundred microliters of RPMI media was added to thetransfection complexes, mixed, and added directly to the cells. Thecells were incubated with the transfection complexes for 3 hours at 37°C. The transfection complexes were then removed, the cells washed withPBS, and the media replaced with HEK, SY5Y or CHO complete media, asappropriate for the cell type. The cells were then incubated at 37° C.For experiments performed in T-75 flasks, all reagent volumes wereincreased 6-fold.

On day 2, the cells were harvested by trypsinization, counted andreplated on PDL-coated plates. For cell calcium measurements, cells werereplated in a 384-well plate at a density of 40,000 cells/well. Forwestern blot or RT-PCR analysis, cells were replated in a 6-well dish ata density of 1,000,000 cells/well. On day 3, cells were assayed forcytosolic calcium levels, as described below. Cell lysates weregenerated to analyze protein levels by western blot, and RNA wasisolated to analyze message levels by RT-PCR, as described below.

B. Analysis of STIM1 Gene Silencing by RT-PCR and Western Blot

1. RT-PCR Analysis

To confirm RNAi-mediated silencing of STIM1, RNA was isolated using anRNeasy™ kit from Qiagen, and RT-PCR used to monitor STIM1 mRNA levels. Areduction in STIM1 mRNA levels in HEK293 or SH-SY5Y cells treated witheither of huSTIM1-1140 or huSTIM1-1414 siRNAs, relative to controlcells, was observed. Likewise, a reduction in STIM1 mRNA in CHO cellstreated with either rSTIM1-114 or rSTIM1-614 siRNAs, relative to controlcells, was observed. For further assays, the huSTIM1-1140 siRNA was usedfor RNAi in human cells, and rSTIM1-114 siRNA was used for RNAi in CHOcells.

2. Western Blot Analysis

For analysis of STIM1 protein levels, cells were lysed in a suitablevolume of lysis buffer (50 mM HEPES, (pH 7.2), 150 mM NaCl, 10%glycerol, 1% TritonX-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mMNa-pyrophosphate and Complete Protease inhibitor (Roche)). Lysates wereincubated on ice for 5 minutes, collected, and centrifuged at 13,000 rpmfor 10 minutes in a microfuge. Supernatants were transferred to a freshtube and protein concentration determined using the BCA Protein Assaykit (Pierce) using BSA as a standard. Cell extracts were loaded onSDS-PAGE gels and subjected to western blot analysis using an anti-STIM1antibody (BD Biosciences).

The results of the western blot analysis indicated that STIM1 proteinlevels were significantly reduced in STIM1 siRNA-treated cells relativeto control cells.

C. Measurement of Cytosolic Calcium

Control cells and cells subjected to RNAi for the silencing of STIM1were analyzed for possible effects of gene silencing on cytosoliccalcium using a fluorescent-based assay.

1. Evaluation of Thapsigargin-Induced Store-Operated Calcium Entry

Cells plated in 384-well plates were loaded for 45 min with FLUO-4-AM (5μM) in a Hanks-buffered salt solution. Cells were washed and placed in anominally Ca²⁺- and Mg²⁺-free Hanks solution. One minute later, 1 μMthapsigargin (Tg) was added to inhibit the ER Ca²⁺ pump and dischargeintracellular Ca²⁺ stores. Fifteen minutes after addition of Tg,store-operated calcium entry was initiated by adding external Ca²⁺ to afinal concentration of 1.8 mM. The calcium response was monitored for afurther 10-15 minutes. Calcium levels were monitored throughout theassay using a FLIPR384 (Molecular Devices fluorimetric imaging platereader for high throughput screening). Analysis of the data revealed asignificant reduction in thapsigargin-induced store-operated calciumentry in HEK293, SH-SY5Y and CHO cells treated with STIM1 siRNA,relative to control cells. A small but variable reduction inthapsigargin-induced ER calcium release was also observed in someexperiments (see FIG. 2). The above experiment was repeated to assessthapsigargin-induced store-operated barium entry, as a surrogate forstore-operated calcium entry. The procedure was identical, except thatinstead of adding external Ca²⁺, store-operated barium entry wasinitiated by adding external ^(Ba2+t)o a final concentration of 10 mM.Barium levels were monitored using the FLIPR384. The results indicatedthat thapsigargin-induced store-operated barium entry is reduced incells treated with STIM1 siRNA relative to control cells.

2. Evaluation of Agonist-Induced Store-Operated Calcium Entry

Cells plated in 384-well plates were loaded for 45 min with FLUO-4-AM (5μM) in a Hanks-buffered salt solution. Cells were washed and placed in anominally Ca²⁺- and Mg²⁺-free free Hanks solution. One minute later, themuscarinic receptor agonist methacholine was added to a finalconcentration of 300 μM to deplete ER calcium stores. Five minuteslater, store-operated calcium entry was initiated by adding externalCa²⁺ to a final concentration of 1.8 mM. The calcium response wasmonitored for a further five minutes. Calcium levels were monitoredthroughout the assay using a FLIPR384 (Molecular Devices fluorimetricimaging plate reader for high throughput screening).

Analysis of the data revealed that methacholine-induced store-operatedcalcium entry was reduced in cells treated with STIM1 siRNA relative tocontrol cells. STIM1 siRNA also had a possible effect on re-uptake ofcalcium following ER calcium release, and/or other calcium bufferingmechanisms, as evidenced by a difference in the decay slope aftermethacholine addition in STIM1 siRNA-treated cells compared to controlcells (see FIG. 3).

The above experiment was repeated to assess agonist-inducedstore-operated barium ion entry, as a surrogate for store-operatedcalcium entry. The procedure was identical, except that instead ofadding external Ca²⁺, store-operated barium entry was initiated byadding external Ba²⁺ to a final concentration of 10 mM. Barium levelswere monitored using the FLIPR384. The results indicated thatmethacholine-induced store-operated barium entry is reduced in cellstreated with STIM1 siRNA relative to control cells.

Example 3

In Vitro Screening for Agents that Modulate Intracellular Calcium Levels

This Example describes fluorescence-based assays that were used forscreening for agents that modulate intracellular calcium.

A. Assay Protocol

SH-SY5Y human neuroblastoma cells plated in 384-well plates were loadedfor 45 min with FLUO-4-AM (2 μM final concentration) in a Hanks-bufferedsalt solution. Cells were washed and placed in a nominally Ca²⁺- andMg²⁺-free Hanks solution. One minute later, a test agent or vehicle wasadded. After a 15 minute incubation period, 1 μM thapsigargin (Tg) wasadded to inhibit the ER Ca²⁺ pump and discharge intracellular Ca²⁺stores. Fifteen minutes after addition of Tg, store-operated calciumentry was initiated by adding external Ca²⁺ to a final concentration of1.8 mM and the cells monitored for a further 10-15 minutes. Calciumlevels were monitored throughout the assay using a FLIPR384 (MolecularDevices fluorimetric imaging plate reader for high throughputscreening).

In an alternative screening assay procedure, one minute after washingout the FLUO-4-AM, 1 μM Tg was added to the SH-SY5Y cells. Fifteenminutes after addition of Tg, test compound or vehicle was added,followed by another 15 minute incubation in Ca²⁺-free buffer.Store-operated calcium entry was then initiated by adding external Ca²⁺to a final concentration of 1.8 mM and the response monitored for afurther 10-15 minutes.

A similar screening assay procedure was used with HEK293 and CHO cells.The screening assay can alternatively use external Ba2+ (finalconcentration of 10 mM) in place of external Ca²⁺. In this case,thapsigargin-induced store-operated Ba2+ entry serves as a surrogate forstore-operated Ca²⁺ entry.

B. Data Analysis

The kinetic data from the FLIPR384 were analyzed and then stored in arelational database (ActivityBase; IDBS). Ten quantitative parameterswere calculated that define various aspects of the store-operatedcalcium entry response. These parameters are as follows:

Mean Basal: basal fluorescence (relative fluorescence units, RFU)readings averaged over 30 seconds prior to addition of Ca²⁺ to initiatestore-operated calcium entry

Up slope: linear regression of the increase in RFU from 2 to 60 secafter addition of Ca²⁺

Up rate constant (Up K): the rate constant derived from first-orderassociation of RFUs from 2 seconds to peak response

Peak: the peak RFU (single point) achieved after addition of Ca²⁺ Timeto peak: the time at which the peak RFU is achieved

Peak/Basal: the difference between peak and mean basal RFU

Decay slope: linear regression of the decrease in RFU from the peak tothe end of the measurement period

Decay rate constant (Decay K): the rate constant derived fromfirst-order decay of RFUs from the peak to the end of the measurementperiod.

Area under the curve (AUC): area under the curve from the addition ofCa²⁺ to the end of the measurement period.

Combinations of these parameters were queried to identify agents thatproduce at least a 50% change from vehicle control. Active agentsidentified from these queries were retested under identical conditionsto confirm their activity. Agents with confirmed activity were thenanalyzed for concentration-dependent effects, and subsequently, thoseagents displaying concentration-dependent effects were categorized asagents that modulate intracellular calcium.

Example 4

Characterization of cDNAs Encoding Rodent Reference STIM1s

This Example describes the procedure used to clone and sequence cDNAencoding Chinese hamster and rat STIM1 partial sequences and referenceSTIM1.

To clone Chinese hamster STIM1, mRNA was isolated from CHO cells. cDNAwas generated from the isolated mRNA using random hexamer primers. PCRprimers were designed based on the human STIM1 cDNA sequence to amplifyoverlapping fragments of human STIM1 cDNA. PCR reactions were performedon the CHO cell cDNA using these primers. The resulting PCR productswere then sequenced. The overlapping nucleotide sequences of the PCRproducts were assembled to yield a partial hamster STIM1 cDNA sequencecorresponding to SEQ ID NO: 95 and encoding SEQ ID NO:96. To clone ratSTIM 1, mRNA was isolated from RBL 2H3 rat mast cells. By a similarprocedure as described above, a partial rat STIM1 cDNA sequence (SEQ IDNO:97) was obtained encoding SEQ ID NO: 98.

To construct reference STIM1, the partial hamster sequence, nucleotides21-2019 of SEQ ID NO:51 was extended with Rattus norvegicus chromosome 1WGS supercontig sequence. This partial hamster STIM1 nucleotide sequenceencodes amino acids 8-673 of SEQ ID NO:52. The nucleotides encoding theN- and C-termini of rat STIM1 were predicted from BLAST alignments ofthe 5′ and 3′ regions, respectively, of cloned hamster STIM1 with theRattus norvegicus chromosome 1 WGS supercontig sequence having GenBankAccession No. NW_(—)043388. The complete reference STIM1 nucleotidesequence corresponds to SEQ ID NO:51 and encodes SEQ ID NO:52.

Example 5

Evaluation of Human STIM2 in Intracellular Calcium Modulation

The effect of RNAi-mediated knockdown of STIM2 on store-operated calciumentry was examined, essentially as described in Example 2. siRNAsspecific for STIM2 were designed and tested for message reduction byRT-PCR. Store-operated calcium entry was then evaluated in HEK293 cellstransfected with either a control siRNA, an siRNA targeting STIM1 or ansiRNA targeting STIM2. Knockdown of STIM1 by RNAi decreased both Ca²⁺and Ba²⁺ entry into cells following thapsigargin-induced storedepletion, as described in Example 2. In contrast, there was no effecton either Ca²⁺ or Ba²⁺ entry in cells treated with the siRNA for STIM2.

Example 6 Specificity of Effect of STIM1 Modulation on Store-OperatedCalcium Entry

STIM1 siRNAs were transfected into SH-SY5Y cells and assayed forstore-operated calcium entry (SOCE), Ca²⁺ entry mediated throughvoltage-gated calcium channels (VGCCs), and membrane potential. SH-SY5Ycells express both N- and L-type VGCCs that can be activated by KCl.Cells treated with the STIM1 siRNA displayed a reduction in SOCE to lessthan 50% of control levels, similar to what was observed in Example 2.To assess effects on Ca²⁺ entry mediated by VGCCs, Fluo-4-loaded cellsbathed in a Ca²⁺-containing buffer were challenged with increasingconcentrations of external KCl to depolarize cells and activate VGCCs.

RNAi mediated knockdown of STIM1 had no significant effect on the peakof the KCl induced Ca²⁺ signal, indicating that knockdown of STIM1 doesnot have a general effect on Ca²⁺ entry mechanisms.

The effect of STIM1 knockdown on membrane potential was also examined inSH-SY5Y cells, under conditions similar to those used to measure VGCCactivity. Control cells and cells transfected with STIM1 siRNA wereloaded with the membrane potential sensitive dye, DiBAC. Cells were thenwashed in calcium free buffer and then challenged with increasingconcentrations of KCl.

STIM1 knockdown produced no detectable effect on the resting membranepotential or the response to depolarization by KCl, indicating that theeffect of STIM1 RNAi on SOCE was not due to a collapse in membranepotential. Thus, STIM1 modulation is specific for store-operated calciumentry.

Example 7 Evaluation of Effect of Recombinant Expression of Human STIM1on Intracellular Calcium

Generation of Stable Cell Lines Overexpressing Human STIM1.

A human STIM1 expression construct was prepared by inserting the 2.7-kbBamH1 fragment from IMAGE clone 4899542 (Research Genetics/Invitrogen)(SEQ ID NO: 94) into the BamH1 site of the pcDNA3.1/Zeo expressionvector (Invitrogen Corp., California). The pcDNA3.1/Zeo expressionvector contains a CMV promoter, T7 promoter priming site, multiplecloning site, BGH reverse priming site, BGH polyadenylation signal, f1origin, SV40 promoter and origin, EM7 promoter, a zeocin resistancegene, SV40 polyadenylation sequence, pUC origin, bla promoter andampicillin (bla) resistance gene. SEQ ID NO: 94 contains the completehuman STIM1 coding sequence as well as 49 nucleotides of 5′ untranslatedsequence and 586 nucleotides of 3′ untranslated sequence. SEQ ID NO: 94also contains a silent polymorphism (CCC->CCT) relative to SEQ ID NO: 3in the codon that encodes PRO675. Plasmid clone pcDNA[STIM1/542-5] wasconfirmed by sequencing.

For stable cell lines overexpressing STIM1, pcDNA[STIM1/542-5] wastransfected into 3×10⁵ HEK293 cells using Effectene (QIAGEN)transfection reagent following the manufacturer's protocol. Forty-eighthours after transfection, cells were harvested and replated underselection with 250 μg/ml Zeocin (Invitrogen). Colonies were selected,amplified, and evaluated for expression of STIM1 mRNA and protein byRT-PCR and western blot, respectively. Limiting dilution of the STIM1overexpressing colonies generated individual clones.

Evaluation of Store-Operated Calcium Entry.

Multiple subclones were isolated that expressed elevated levels of STIM1protein, one of which (referred to as HEK[STIM1]) expresses STIM1 atlevels greater than 100-fold compared to HEK-Zeo controls. In multiplesubclones, overexpression of STIM1 resulted in constitutive activationof a Ca²⁺ entry pathway in the absence of thapsigargin or agonistinduced store depletion. An example of this effect is shown for cloneHEK[STIM1] (FIG. 4 panel A). Thus, when STIM1-overexpressing cells weretreated with vehicle (DMSO) instead of thapsigargin in Ca²⁺-free buffer,readdition of external Ca²⁺ resulted in Ca²⁺ entry with kineticsqualitatively similar to the kinetics of thapsigargin-dependent Ca²⁺entry in control cells.

In addition to activation of SOCE in the absence of store depletion,stable overexpression of STIM1 enhanced SOCE activated by thapsigargin(FIG. 4 panel B). However, a significant decrease in the amplitude andkinetics of the thapsigargin-mediated release of calcium from theinternal stores was also observed. Calcium entry was also increased whenstores were depleted by the muscarinic agonists carbachol ormethylcholine. Thus, stable overexpression of STIM1 reduces thethapsigargin-mediated discharge of calcium from the internal stores andenhances SOCE when elicited by either thapsigargin or agonists todeplete the internal calcium stores. To exclude clonal variations in theresults, similar experiments were performed with a second clone withsimilar results. These results were dependent on STIM1 overexpression,because overexpression of huAPP, an unrelated single pass transmembraneprotein, had no detectable effect on TG-dependent or TG-independent SOCEin HEK293 cells.

Profiles of Ca²⁺, Sr²⁺ and Ba²⁺ entry. The relative cation permeabilitywas assessed in HEK-Zeo and in HEK[STIM1] cells. Cells were loaded withFluo-4 and treated with thapsigargin to discharge intracellular Ca²⁺stores (HEK-Zeo cells), or vehicle-treated (both HEK-Zeo and HEK[STIM1]cells). After 15 minutes, 10 mM BaCl₂ (final concentration), 10 mM SrCl₂(final concentration), or 1.8 mM CaCl₂ (final concentration) was addedto elicit cation influx. To quantify the rate of cation influx, thefirst 30 seconds of data following cation addition was analyzed bylinear regression.

In HEK-Zeo cells, treatment with thapsigargin to deplete intracellularCa²⁺ stores resulted in a greater increase in the initial rate of Ca²⁺and Sr²⁺ entry compared to Ba²⁺ entry. To test whether the cation entrypathway activated by STIM1 overexpression in HEK293 cells is similar tothe endogenous store-operated pathway, the profile of Ca²⁺-, Ba²⁺, andSr²⁺-entry was assessed in the absence of store-depletion. HEK[STIM1]cells displayed a similar pattern of response compared to HEK-Zeo cellsstimulated with thapsigargin, wherein the initial rates of Ca²⁺ and Sr²⁺entry were markedly increased compared to Ba²⁺.

Thus, the profile of STIM1-induced Ca²⁺, Sr²⁺, and Ba²⁺ entry resemblesthe profile of endogenous store-operated cation entry in response tostore-depletion.

Example 8 Modulation of Intracellular Calcium by a SOCE Inhibitor inSTIM1-Overexpressing Cells

Store-operated calcium entry is sensitive to the inhibitor2-aminoethoxydiphenyl borate (2-APB). To test whether the Ca²⁺ entrypathway constitutively activated by STIM1 overexpression ispharmacologically similar to endogenous SOCE, HEK[STIM1] cells werepre-incubated with increasing doses of 2-APB and STIM1-induced Ca²⁺entry was measured. Thapsigargin-mediated store depletion of bothHEK-Zeo control cells and HEK[STIM1] cells followed by readdition ofexternal calcium resulted in inhibition by 2-APB with similar IC₅₀values of 11.8 μM and 10.5 μM, respectively. Treatment of HEK[STIM1]cells with 2-APB and examining calcium entry in the absence of storedepletion resulted in a biphasic effect of 2-APB on calcium entry. Theconstitutive calcium entry was inhibited with an IC₅₀ value of 10.8 μM,similar to that reported for endogenous SOCE. However, at lowerconcentrations of 2-APB, calcium entry was potentiated. The ability toboth potentiate and inhibit calcium entry is a property of 2-APB thathas previously been shown to occur with the calcium release activatedcalcium (CRAC) channel.

Thus, overexpression of STIM1 in HEK293 cells confers a CRAC-likeproperty to constitutive Ca²⁺ entry measured in HEK293 cells.Accordingly, assays to identify agents that modulate intracellularcalcium can optionally be performed in cells overexpressing STIM1 in theabsence of intracellular calcium depletion protocols.

Example 9 Effect of STIM1 RNAi on Degranulation, Cytokine Release andCCE in Mast Cells

The effect of STIM4 modulation on degranulation, cytokine release andCCE in mast cells was assessed. RBL-2H3 rat mast cells wereelectroporated with siRNA STIM1-r114 (SEQ ID NO:91), using the Bio-RadGene Pulser electroporator. Three days after transfection, cells wereanalyzed for levels of rat STIM1 protein as well as for effects onbiological properties of mast cells.

To assess STIM1 protein levels, protein extracts were generated andresolved by SDS-PAGE and analyzed by western blot. Using a monoclonalantibody to STIM1 (anti-GOK), which cross-reacts with rat STIM1, levelsof STIM1 were knocked down greater than 50% relative to control samples(cells transfected with a non-silencing, scrambled siRNA).

To assess degranulation and cytokine release, cells were plated andstimulated with 20 nM thapsigargin/20 nm TPA for 20 hr. Media wascollected and assayed for the release of the inflammatory mediatorβ-hexosaminidase or for the release of the cytokine TNF-α. Theβ-hexosaminidase enzymatic assay was performed by adding 200 μl 1 mMp-nitrophenyl-acetyl-glucosamide substrate (Sigma #N9376) in 0.05M NaCitrate (pH 4.5) to 50 μl of conditioned media, incubating for 60′ at37° C., then adding 500 μl 0.05M Na Carbonate, 0.05M Na bicarbonate pH10.5, mixing thoroughly, and reading the absorbance at 405 nm in aBiorad plate reader. The TNF-α release assay was performed using the RatTumor Necrosis Factor-α Ultrasensitive ELISA Kit from Biosource. Cellstransfected with STIM1-r114 siRNA showed a decrease in bothβ-hexosaminidase activity and TNF-α release relative to control cells.

To assess calcium entry, cells were plated in a 384-well plate, loadedwith the calcium sensitive dye Fluo-4-AM, placed in a FLIPR384 andassayed for calcium entry. Cells were stimulated with 1 μM TG in calciumfree buffer for 15 minutes and then 2 mM calcium was added. Cellstreated with STIM1-r114 siRNA showed a decrease in calcium entry uponstore-depletion protocols, relative to control cells.

Thus, RNAi-mediated knockdown of STIM1 in rat mast cells reducesTG-induced calcium entry as well as TG/TPA-induced degranulation andcytokine release.

Example 10 Effect of STIM1 RNAi on Cytokine Secretion

Cytokine Secretion in PHA-Stimulated T cells. In Jurkat T cells, T cellreceptor (TCR) stimulation leads to activation of the CRAC channel andsubsequent gene expression and cytokine release. If STIM1 plays animportant role in CRAC channel function, then one would predict thatcytokine release in response to TCR stimulation with PHA would beaffected by knockdown of STIM1 in Jurkat T cells.

To test this prediction, a stable Jurkat T cell line expressing a shorthairpin siRNA to suppress expression of STIM1 was generated as follows.Oligonucleotides corresponding to the STIM1-1140 siRNA (sense oligo,5′-GAT CCC GGC TCT GGA TAC AGT GCT CTT CAA GAG AGA GCA CTG TAT CCA GAGCCT TTT TTG GAA A-3′ SEQ ID NO: 99; antisense oligo, 5′-AGC TTT TCC AAAAAA GGC TCT GGA TAC AGT GCT CTC TCT TGA AGA GCA CTG TAT CCA GAG CCG G-3′SEQ ID NO: 100) were annealed and ligated into pSilencer 2.1-U6 neo(Ambion) according to the manufacturer's protocol. Correct inserts wereverified by DNA sequencing. For the control, a non-silencing scrambledsiRNA (Negative Control oligo; Ambion) was generated as above. 2×10⁶Jurkat T cells were electroporated with 5 μg plasmid using a Bio-RadGene Pulser (140V, 30 msec, 4 pulses). Two days after transfection,cells were harvested and resuspended in media containing G-418 selectionat a concentration of 1×10⁴ cells/ml. 1 ml of cells/well was plated in24-well plates. Stable pools of cells expressing the short hairpin loopconstructs were characterized for STIM1 expression by western blot, andone of these pools was designated Jurkat clone 4A5. Stable pools ofJurkat cells expressing scrambled siRNA were also generated (JurkatNegative Control, clone 2A4).

To measure IL-2 secretion from Jurkat T cells, cells were plated in a 96well plate at a density of 1.5×10⁵ cells/well. Cells were stimulatedwith 2.5 μg/ml PHA lectin+80 nM TPA for 20 hours. The media wascollected and analyzed for IL-2 levels by ELISA (BioSource) according tothe manufacturer's protocols.

Either Jurkat clone 4A5 or Jurkat Negative Control pools were stimulatedwith PHA/TPA, a combination of lectin and phorbol ester used forstimulating Jurkat cells for cytokine release studies. IL-2 secretion incontrol cells required PHA/TPA stimulation. In cells expressing theSTIM1-1140 short hairpin siRNA, secretion of IL-2 upon stimulation wascompletely blocked.

To assess the effect of STIM1 knockdown on calcium entry, cells wereloaded with the calcium sensitive dye fluo4-AM and stimulated with PHA.STIM1 knockdown cells showed a delay in calcium entry upon stimulationas well as a sustained reduction in the level of cytosolic calciumthroughout the stimulation protocol. Thus, modulation of STIM1 levels inJurkat T-cells affects both PHA-dependent calcium entry and cytokinerelease.

Cytokine Secretion in T Cells Activated by Other Stimuli.

IL-2 stimulation can be elicited by several stimuli that act throughdifferent pathways. anti-OKT3 activates cells through the CD3 receptor,whereas thapsigargin (TG), ionomycin and TPA activate cells throughnon-TCR mechanisms. PHA activates cells through the TCR, as describedabove.

Negative control cells and clone 4A5 cells were plated in a 96-wellplate and stimulated with either vehicle (0.005% DMSO)+80 nM TPA, 1.25μg/ml anti-OKT3 +80 nM TPA, 2.5 μg/ml PHA+80 nM TPA, 0.5 μM TG+80 nMTPA, or 1 μM ionomycin for 20 hours. IL-2 secretion was measured byELISA (BioSource).

In control cells, there was a modest level of IL-2 secretion byanti-OKT3/TPA stimulation and a significant level of IL-2 release byPHA/TPA, TG/TPA, and ionomycin/TPA. In clone 4A5 Jurkat cells (whereSTIM1 protein levels were lower) there were barely detectable levels ofIL-2 secretion upon stimulation by any of these agents.

To examine stimulus-induced calcium entry in these cells, eithernegative control cells or clone 4A4 cells were loaded with Fluo-4-AM andanalyzed for calcium entry as described above. Cells were stimulatedwith vehicle (0.005% DMSO), 1.25 μg/ml anti-OKT3, 0.5 μM TG, or 2.5μg/ml PHA in calcium containing buffer. As described above, cellsstimulated with PHA showed an inhibition in the rate of calcium entry aswell as a sustained reduction in the level of calcium. The results uponPHA stimulation were also seen in single cell imaging experiments. Inimaging experiments the delay in calcium entry was much greater thanseen in the FLIPR³⁸⁴ experiments. A similar decrease in the level of thesustained calcium plateau upon stimulation was also seen.

When stimulated with anti-OKT3, STIM1 knockdown cells (clone 4A5) showedan increase in the amplitude of calcium entry, but no increase in therate of calcium entry relative to control cells. STIM1 knockdown hadvery little effect on the rate of calcium entry or the sustained levelin TG stimulated cells.

Thus, STIM1 knockdown reduces IL-2 secretion under some conditions whereCCE entry is not affected. This suggests that modulation of STIM1 canhave effects on CCE-independent gene expression or secretion, includingspecific effects on CCE-independent IL-2 expression or IL-2 secretion.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. (canceled)
 2. A method of identifying an agent that modulatesintracellular calcium, comprising: contacting one or more test cells ora portion thereof comprising a stromal interacting molecule (STIM)protein or portion thereof; assessing the effect(s) of the test agent onintracellular calcium; and identifying a test agent as an agent thatmodulates intracellular calcium if it has an effect on intracellularcalcium. 3-36. (canceled)
 37. A method of identifying an agent thatmodulates intracellular calcium, comprising: assessing the effects of atest agent on intracellular calcium, wherein the test agent modulates anactivity of, an interaction of, the level of or binds to or interactswith a STIM or STIM-like protein; and identifying a test agent as anagent that modulates intracellular calcium if it has an effect onintracellular calcium. 38-74. (canceled)
 75. A method of identifying anagent that modulates intracellular calcium, comprising: assessing theeffects of a test agent on a stromal interacting molecule (STIM) orSTIM-like protein, or portion thereof, wherein the test agent modulatesintracellular calcium; and identifying a test agent as an agent thatmodulates intracellular calcium if it has an effect on a STIM orSTIM-like protein, or portion thereof. 76-100. (canceled)
 101. A system,comprising: a cell, or portion thereof, comprising one or moreheterologous STIM of STIM-like proteins, or a portion thereof and/orheterologous nucleic acid encoding one or more STIM or STIM-likeproteins, or a portion thereof; and an agent that provides for reductionof calcium levels in an intracellular calcium store.
 102. (canceled)103. A system, comprising: a cell, or portion thereof, comprising one ormore heterologous STIM of STIM-like proteins, or a portion thereof,and/or heterologous nucleic acid encoding one or more STIM of STIM-likeproteins, or a portion thereof; and a molecule used in monitoring ormeasuring calcium and/or calcium movement. 104-116. (canceled)
 117. Amethod of identifying a molecule involved in modulating intracellularcalcium, comprising: assessing the effect of a test molecule onintracellular calcium, wherein the test molecule interacts with a STIMor STIM-like protein, or portion thereof; and identifying a testmolecule as a molecule involved in modulating intracellular calcium ifit has an effect on intracellular calcium. 118-138. (canceled)
 139. Amethod of modulating intracellular calcium, comprising modulating in acell, or portion thereof, one or more proteins, or nucleic acid encodingone or more proteins, that is (are) at least about 45% homologous to theprotein encoded by Drosophila gene CG9126 and/or a stromal interactingmolecule (STIM) or STIM-like protein over at least about 52% of theprotein; thereby modulating intracellular calcium in a cell or portionthereof. 140-159. (canceled)
 160. A method for identifying an agent thatmodulates intracellular calcium, comprising: assessing the effects of atest agent on intracellular calcium of a test cell or portion thereof;and identifying a test agent as an agent that modulates intracellularcalcium if it has an effect on intracellular calcium of the test cell;wherein: the test cell or a portion thereof comprises a polymorphic formof one or more STIM or STIM-like proteins and/or a polymorphic form of agene or nucleic acid encoding one or more STIM or STIM-like proteins;and intracellular calcium of the test cell differs from intracellularcalcium of a cell, or portion thereof, that contains a wild-type form ofthe protein(s).
 161. (canceled)
 162. (canceled)
 163. A method foridentifying an agent that modulates intracellular calcium, comprising:assessing the effects of a test agent on intracellular calcium of a testcell, or portion thereof, that exhibits calcium dyshomeostasis; andidentifying a test agent as an agent that modulates intracellularcalcium if it has an effect on intracellular calcium of the test cell,wherein: the test agent modulates an activity of, an interaction of, thelevel of or binds to or interacts with a STIM or STIM-like protein.164-190. (canceled)
 191. A method for identifying an agent for treatingor preventing a disease or disorder involving an alteration inintracellular calcium, comprising: assessing the effects of a test agenton intracellular calcium of a test cell or portion thereof; andidentifying a test agent as an agent for treating or preventing adisease or disorder if it has an effect on intracellular calcium of thetest cell; wherein: the test cell is a cell of an animal comprising apolymorphic form of one or more STIM or STIM-like proteins and/or apolymorphic form of a gene, or portion thereof, or nucleic acid encodingone or more STIM or STIM-like proteins; and a cell comprising thepolymorphic form of the one or more proteins and/or the polymorphic formof a gene, or portion thereof, or nucleic acid encoding the one or moreproteins exhibits calcium dyshomeostasis.
 192. (canceled) 193.(canceled)
 194. A method for identifying an agent for treating orpreventing a disease or disorder involving an alteration inintracellular calcium, comprising: assessing the effects of a test agenton a phenotype of an organism; and identifying a test agent as an agentfor treating or preventing a disease or disorder if it has an effect ona phenotype of the organism, wherein: the test agent modulates anactivity of, an interaction of, the level of or binds to or interactswith a STIM or STIM-like protein; and the organism comprises one or morecells that exhibit calcium dyshomeostasis.
 195. (canceled)
 196. A methodfor identifying an agent for treating or preventing a disease ordisorder involving an alteration in intracellular calcium, comprising:assessing the effects of a test agent on a phenotype of an organism; andidentifying a test agent as an agent for treating or preventing adisease or disorder if it has an effect on a phenotype of the organism,wherein: the test agent modulates an activity of, an interaction of, thelevel of or binds to or interacts with a STIM or STIM-like protein; andthe organism exhibits a phenotype associated with a disease or disorderthat involves or is characterized at least in part by calciumdyshomeostasis.
 197. (canceled)
 198. A method for identifying an agentfor treating or preventing a disease or disorder, comprising: assessingthe effects of a test agent on a phenotype of an organism; andidentifying a test agent as an agent for treating or preventing adisease or disorder if it has an effect on a phenotype of the organism,wherein: the test agent modulates an activity of, an interaction of, thelevel of or binds to or interacts with a STIM or STIM-like protein; andthe organism exhibits a phenotype associated with a disease or disorderselected from the group consisting of an immune system-related disease,a disease involving inflammation, a renal system disease, aneurodegenerative disease, pain and liver disease. 199-209. (canceled)210. A method for treating or preventing a disease or disorder,comprising: modulating in a subject having a disease or disorder, or atrisk for developing a disease or disorder, one or more proteins, and/ora gene or nucleic acid encoding one or more proteins, that is (are) atleast about 45% homologous to the protein encoded by Drosophila geneCG9126 and/or a stromal interacting molecule (STIM) or STIM-like proteinover at least about 52% of the protein; wherein the disease or disorderinvolves, or is characterized at least in part by: (1) alteredintracellular calcium, altered intracellular calcium regulation orcalcium dyshomeostasis or dysregulation and/or (2) an alteration ordefect in, or aberrant functioning of, a cellular process which relieson or is regulated by intracellular calcium. 211-235. (canceled)
 236. Anisolated nucleic acid molecule, comprising a sequence of nucleotidesencoding a rodent reference STIM1 polypeptide or a mature referenceSTIM1 polypeptide, wherein the sequence of nucleotides is selected fromthe group consisting of a) a sequence of nucleotides that encodes apolypeptide comprising amino acids 23-685 set forth in SEQ ID NO: 52; b)a sequence of nucleotides that encodes a polypeptide comprising theamino acids set forth in SEQ ID NO: 52; c). a sequence of nucleotidesthat encodes a polypeptide comprising the amino acids set forth in SEQID NO: 96; and d) a sequence nucleotides that encodes a polypeptidecomprising the amino acids set forth in SEQ ID NO:
 98. 237-242.(canceled)
 243. An isolated oligonucleotide, comprising at least 17contiguous nucleotides of SEQ ID NO:51, wherein the contiguousnucleotides include a position selected from the group consisting ofpositions 15, 63, 69, 84, 103, 108, 112, 120, 150, 183, 201, 207, 210,213, 240, 300, 303, 312, 330, 357, 402, 441, 474, 570, 621, 660, 697,738, 783, 795, 861, 873, 874, 895, 951, 1051, 1062, 1107, 1200, 1224,1228, 1278, 1299, 1392, 1395, 1452, 1580, 1652, 1654, 1675, 1747, 1749,1173, 1854, 1855, 1881, 1884, 1888, 1896, 2001 and 2025 of SEQ ID NO:51.244. An isolated oligonucleotide that specifically hybridizes to SEQ IDNO:51, but does not specifically hybridize to SEQ ID NO:3, SEQ ID NO:9or SEQ ID NO:82.
 245. An isolated polypeptide, selected from the groupconsisting of: a) amino acids 23-685 of SEQ ID NO:52 (reference STIM1)or a sequence of amino acids with no greater than 13 amino acidsubstitutions relative to SEQ ID NO:52; b) SEQ ID NO:96; and c) SEQ IDNO:98.
 246. A method of identifying an agent that modulatesintracellular calcium, comprising: contacting, with a test agent, one ormore test cells overexpressing a mammalian STIM1 protein or a portionthereof that retains at least one biological activity of a mammalianSTIM1 protein; assessing the effect of the test agent on intracellularcalcium; and identifying a test agent as an agent that modulatesintracellular calcium if it has an effect on intracellular calcium. 247.A method of identifying an agent that modulates intracellular calcium,comprising: contacting one or more test cells with a test agent, whereinthe test agent modulates an activity of, modulates an interaction of ormodulates the level of, or binds to or interacts with a mammalian STIM1protein or a portion thereof that retains at least one biologicalactivity of a mammalian STIM1 protein; assessing the effect of the testagent on intracellular calcium; and identifying a test agent as an agentthat modulates intracellular calcium if it has an effect onintracellular calcium.
 248. (canceled)
 249. A method of identifying anagent that modulates STIM1, comprising: assessing the effect of the testagent on an activity of, an interaction of or the level of a mammalianSTIM1 protein or a portion thereof that retains at least one biologicalactivity of a mammalian STIM1 protein, or on binding to or interactionwith mammalian STIM1 protein or a portion thereof that retains at leastone biological activity of a mammalian STIM1 protein, wherein the testagent modulates intracellular calcium; and identifying an agent thatmodulates the STIM1 protein or portion thereof if it has an effect on anactivity of, an interaction of or the level of STIM1, or binds to orinteracts with the STIM1 protein or portion thereof. 250-255. (canceled)256. A method of identifying an agent that modulates cytokine expressionor secretion, comprising: contacting, with a test agent, one or moretest cells overexpressing a mammalian STIM1 protein or a portion thereofthat retains at least one biological activity of a mammalian STIM1;assessing the effect of the test agent on cytokine expression orsecretion; and identifying a test agent that modulates cytokineexpression or secretion.
 257. A method of identifying an agent thatmodulates cytokine expression or secretion, comprising: contacting oneor more test cells with a test agent, wherein the test agent modulatesan activity of, modulates an interaction of or modulates the level of,or binds to or interacts with a mammalian STIM1 protein or a portionthereof that retains at least one biological activity of a mammalianSTIM1 protein; assessing the effect of the test agent on cytokineexpression or secretion; and identifying a test agent that modulatescytokine expression or secretion.
 258. (canceled)
 259. (canceled)
 260. Asystem, comprising: a cell, or portion thereof, overexpressing one ormore mammalian STIM1 proteins or a portion thereof that retains at leastone biological activity of a mammalian STIM1 protein; and a moleculeused in monitoring, measuring or detecting a calcium-entry mediatedevent. 264-265. (canceled)
 261. (canceled)
 262. (canceled)
 263. Asystem, comprising: a cell, or portion thereof, containing aheterologous nucleic acid molecule comprising one or more mammalianSTIM1 nucleic acid molecules or a portion thereof, wherein theexpression of STIM1 in the cell or portion thereof is reduced; and amolecule for monitoring, measuring or detecting a calcium-entry mediatedevent.
 264. (canceled)
 265. (canceled)